Immobilization of an active agent on a substrate

ABSTRACT

The invention provides methods of immobilizing an active agent to a substrate surface, including the steps of, depositing a primer compound on a substrate, thereby forming a primed substrate, contacting the primed substrate with a solution of a compound including a trihydroxyphenyl group, thereby forming a trihydroxyphenyl-treated primed substrate, and contacting the trihydroxyphenyl-treated primed substrate with a solution of an active agent, thereby immobilizing the active agent on the substrate. Further provided are methods of immobilizing an active agent on a substrate, including the steps of providing a substrate, combining a solution of a compound including a trihydroxyphenyl group with a solution of an active agent, thereby forming a solution of an active agent-trihydroxyphenyl conjugate, and contacting the primed substrate with the solution of the active agent-trihydroxyphenyl conjugate, thereby immobilizing the active agent on the substrate. The invention further provides substrates and medical device or device components with active agents immobilized on the surface thereof.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

The claimed invention was made as a result of activities undertakenwithin the scope of a joint research agreement between Baxter HealthcareCorporation, and Northwestern University.

FIELD OF THE INVENTION

The invention relates generally to the immobilization of an active agenton a substrate. More particularly, the invention relates to methods ofimmobilizing an active agent on a substrate, substrates with activeagents immobilized thereto, and medical devices comprising substrateswith active agents immobilized thereto.

BRIEF DESCRIPTION OF RELATED TECHNOLOGY

Medical devices and medical device components that are used forhemodialysis or other applications that involve contact betweenphysiologic fluids, such as blood, or tissue and the medical device ordevice component are known to become fouled with protein, cell, and/orbacterial deposits from the contact. The deposition of protein from theblood onto medical devices or medical device components is problematicfor a number of materials commonly used as substrates for medicaldevices and medical device components, especially polysulfone,polycarbonate, and silicone. In many cases, the fouling can impairfunction or lead to failure of the medical device. This problem isparticularly significant for extracorporeal blood circuits andcomponents thereof such as the tubing used in a hemodialysis set.

Coating substrates with active agents, for example,antifouling/antimicrobial agents, is known in the art. For example,3,4-dihydroxyphenylalanine (DOPA) has been used to synthesizedihydroxyphenyl containing polymers which can be used as adhesivepolymers which also provide antifouling/antimicrobial coatings, asdescribed in U.S. Pat. No. 7,618,937, and U.S. Patent ApplicationPublication Nos. 2010/0028719, 2009/0123652, 2008/0247984, 2008/0169059,and 2006/0009550. Typically, the polymers derived from DOPA compriseanchor moieties comprised of peptides, such as lysine, copolymerizedwith DOPA, as shown in structure (I) below, which can be costly to massproduce. It is believed that a peptide or peptoid moiety, coupled to theanchor moiety is generally resistant to, or inhibits protein adsorptionor cell fouling of the surfaces onto which the composition is coated orattached.

Alternatively, U.S. Pat. No. 7,622,533 and U.S. Patent ApplicationPublication No. 2010/0197868 describe an adhesive polymer includingpendant DOPA groups or dihydroxyphenyl (DHDP) derivatives attachedthereto to form adhesive polymers capable of binding to a dissimilarsubstrate, as shown in structure (II) below.

However, with both approaches, leaching of the DOPA from the polymer isa significant toxicity concern.

SUMMARY

The invention provides methods of immobilizing an active agent on asubstrate surface, including the steps of, depositing a primer compoundon a substrate thereby forming a primed substrate, contacting the primedsubstrate with a solution of a compound including a trihydroxyphenylgroup, thereby coupling the trihydroxyphenyl group to the substrate toprovide a trihydroxyphenyl-treated primed substrate, and contacting thetrihydroxyphenyl-treated primed substrate with a solution of an activeagent, thereby immobilizing the active agent on the surface thereof. Thecompound including a trihydroxyphenyl group can be a small molecule or apolymer including a trihydroxyphenyl group. The polymer can be a polymerincluding the trihydroxyphenyl group in the backbone of the polymer, oralternatively a polymer including at least one monomer having a pendanttrihydroxyphenyl group.

In a related aspect, the invention further provides methods ofimmobilizing an active agent on a substrate, including the steps ofdepositing a primer compound on a substrate thereby forming a primedsubstrate, combining in solution a compound including a trihydroxyphenylgroup and an active agent, thereby forming a solution of an activeagent-trihydroxyphenyl conjugate, and contacting the primed substratewith the solution of the active agent-trihydroxyphenyl conjugate,thereby coupling the trihydroxyphenyl group of the activeagent-trihydroxyphenyl conjugate to the primed substrate, andimmobilizing the active agent on the surface thereof. The compoundincluding a trihydroxyphenyl group can be a small molecule or a polymerincluding a trihydroxyphenyl group. The polymer can be a polymerincluding the trihydroxyphenyl group in the backbone of the polymer, oralternatively a polymer including at least one monomer having a pendanttrihydroxyphenyl group.

The invention further provides methods of immobilizing an active agenton a substrate surface, including the steps of, depositing a primercompound on a substrate, thereby forming a primed substrate, contactingthe primed substrate with a solution of gallic acid, thereby couplingthe gallic acid to the substrate to provide a gallic acid-treated primedsubstrate, and contacting the gallic acid-treated primed substrate witha solution of an active agent, thereby immobilizing the active agent onthe substrate surface.

In another related aspect, the invention provides substrates having anactive agent immobilized on a surface thereof, the substrate including alayer of a primer compound on the substrate surface, wherein the layerof the primer compound includes a trihydroxyphenyl group coupledthereto, and wherein the trihydroxyphenyl group includes an active agentcoupled thereto and thereby immobilized on the substrate surface. Theactive agent can be coupled to the trihydroxyphenyl group and/or theprimer compound via a linker compound, so as to immobilize the activeagent on the substrate. The compound including a trihydroxyphenyl groupcan be a small molecule or a polymer including a trihydroxyphenyl group.The polymer can be a polymer including the trihydroxyphenyl group in thebackbone of the polymer, or alternatively a polymer including at leastone monomer having a pendant trihydroxyphenyl group.

In another related aspect, the invention provides medical devicesincluding a substrate according to the invention.

Further aspects of the invention may become apparent to those skilled inthe art from a review of the following detailed description, taken inconjunction with the appended claims. While the invention is susceptibleof embodiments in various forms, described hereinafter are specificembodiments of the invention with the understanding that the disclosureis illustrative, and is not intended to limit the invention to specificembodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows x-ray photoelectron survey spectra of polysulfone surfacesmodified with (i) unmodified polysulfone surface, (ii)chitooligosaccharide and gallic acid and (iii) chitooligosaccharide,gallic acid and different concentrations of heparin.

FIG. 2 shows a plot of thrombin conversion (ng/ml) vs. time (minutes)for different substrates relative to a control.

DETAILED DESCRIPTION

The invention provides substrates with an active agent advantageouslyand securely immobilized on a surface thereof and methods for formingsame. The substrates with an active agent immobilized thereto areparticularly advantageous in that they can be produced relativelyinexpensively, particularly relative to prior art substrates coated withadhesive polymers derived from peptide-DOPA copolymers. The substrateswith active agents immobilized thereto can also be particularlyadvantageous in that they demonstrate low toxicity, particularlyrelative to prior art substrates coated using DOPA-based adhesivepolymers.

The invention provides methods of immobilizing an active agent on asubstrate surface, including the steps of, depositing a primer compound(generally including a nucleophilic group) on a substrate, therebyforming a primed substrate, contacting the primed substrate with asolution of a compound including a trihydroxyphenyl group to couple thecompound including a trihydroxyphenyl group to the primer compound,thereby coupling the trihydroxyphenyl group to the substrate to providea trihydroxyphenyl-treated primed substrate, and contacting thetrihydroxyphenyl-treated primed substrate with a solution of an activeagent to couple the active agent to the trihydroxyphenyl group, therebyimmobilizing the active agent on the surface thereof. The methods canfurther include the step of contacting the trihydroxyphenyl-treatedprimed substrate with a solution of a linker compound thereby couplingthe linker compound to the trihydroxyphenyl group and/or the primercompound of the trihydroxyphenyl-treated primed substrate, prior tocontacting the trihydroxyphenyl-treated substrate with the active agent.

As used herein, “immobilizing” or “immobilized” encompasses any ofsecuring, attaching, affixing, connecting, and/or joining, an activeagent to a substrate surface. Immobilization of the active agent to thesubstrate surface can be confirmed using a number of differenttechniques. For example, as demonstrated in the examples, theimmobilization of the active agent can be confirmed by demonstratingthat the activity of the active agent is present using assays known inthe art. The activity of the active agent can be assessed withfunctional assays. For example, a thrombogenic assay can be used todetect anti-thrombogenic agents such as heparin, 4-hydroxycoumarin, andthe like. Further, for example, the active agent may be labeled with afluorescent dye, an isotopic label, or a radiolabel that can be detectedon the substrate when the active agent is immobilized thereto. Thepresence of the active agent can also be determined with surfacespectroscopies such as x-ray photoelectron spectroscopy (XPS), Fouriertransform infrared reflection-absorption spectroscopy (FTIRRAS), andRaman spectroscopy. Further, cationic stains can be used toconfirm/detect the presence of anionic active agents, for example,Alcian blue and Toluidine blue form a complex with anionic active agentssuch as heparin.

As used herein, “coupling” and “couple” encompass any of covalent bondformation, hydrogen bond formation, ionic bond formation (e.g.,electrostatic attraction), and van der Waals interactions, for example,through which the compound including a trihydroxyphenyl group can adsorbto/adhere to/couple to/associate with the primer layer or a linkercompound, and through which the active agent can adsorb to/adhere/coupleto/associate with a compound including a trihydroxyphenyl group or alinker compound.

As used herein, “compound including a trihydroxyphenyl group”encompasses small molecule compounds, polymers includingtrihydroxyphenyl groups, and trihydroxyphenyl-linker conjugates. Thepolymers including trihydroxyphenyl groups include polymers wherein thetrihydroxyphenyl group is in the polymer backbone, and polymersincluding at least one monomer having a pendant trihydroxyphenyl group.

As used herein, “trihydroxyphenyl group” refers to a compound comprisinga phenyl ring substituted with at least three hydroxyls. Thetrihydroxyphenyl group therefore includes compounds comprising a phenylring substituted with three hydroxyls, and even with four hydroxyls.Generally, compounds comprising a phenyl ring substituted with at leastthree hydroxyls are preferred. Compounds comprising a phenyl ringsubstituted with three hydroxyls are advantageous because in addition tothe three hydroxyl groups, such compounds have three potential sites ofreactivity available, which sites can be selected from but are notlimited to unsubstituted carbons and reactive groups. For example, twounsubstituted carbons and/or reactive groups can couple the compoundincluding a trihydroxyphenyl group to a primer compound and an activeagent, a primer compound and a linker compound, or to two additionalcompounds including a trihydroxyphenyl group via sites of reactivity onthe additional trihydroxyphenyl groups (i.e., resulting in polymerformation). A compound with a third site of reactivity, in addition tothe coupling that can be done with two sites of reactivity, canadvantageously also couple to a linker compound, an active agent, oranother compound including a trihydroxyphenyl group, and can beparticularly advantageous for crosslinking of polymers includingtrihydroxyphenyl groups. Further, without intending to be bound by anyparticular theory, it is believed that compounds comprising a phenylring substituted with three hydroxyls are advantageous over compoundshaving one or two hydroxyls because typically the unsubstituted carbonson compounds comprising three hydroxyls are relatively more reactive.For example, as the number of hydroxyls on the phenyl ring increases,the rate of oxidation generally increases and thus it is typicallyrelatively easier for compounds containing trihydroxyphenyl groups toform quinone-like species than corresponding compounds have phenylgroups substituted with only one or two hydroxyls. Consequently,compounds comprising a phenyl ring substituted with at least threehydroxyls typically have unsubstituted carbons that are relatively morereactive than unsubstituted carbons on corresponding compounds havingphenyl groups substituted with only one or two hydroxyls.

As used herein, “sites of reactivity” or “reactive sites” on thecompound including a trihydroxyphenyl group do not refer to the hydroxylmoieties themselves, but refer to any other site on the compoundincluding a trihydroxyphenyl group through which an active agent, primercompound, linker compound, or additional compound including atrihydroxyphenyl group can couple to the compound including atrihydroxyphenyl group. For example, sites of reactivity can includeunsubstituted carbons and reactive groups which can include, but are notlimited to, carboxyls, carboxylates, amides, acyl halides, aldehydes,ketones, and esters Of course, the hydroxyl moieties of the compoundincluding a trihydroxyphenyl group can also demonstrate reactivity, forexample, by forming ester linkages with primer compounds having acidside chains such as poly(methacrylic acid), poly(acrylic acid),poly(glutamic acid), and poly(malic acid).

As used herein, “polymer” encompasses any compound with two or morerepeat units, for example, dimers, trimers, and higher oligomers. Therepeat units can be the same such that a homopolymer is provided, ordifferent such that a copolymer is provided.

As used herein, “active agent” encompasses active agents (includingthose specifically mentioned herein) and active agent-linker conjugates.

As used herein, “linker compound” encompasses any compound that has atleast two end groups such that the linker compound can couple to andthereby connect two separate molecules. For example, the linker compoundcan couple to either a reactive group and/or an unsubstituted carbon ofthe trihydroxyphenyl group through a first end group and to apolymerizable moiety through a second end group, so as to form apolymerizable monomer. Alternatively, the linker compound can couple toeither a reactive group and/or an unsubstituted carbon of thetrihydroxyphenyl group through a first end group and to an active agentthrough a second end group so as to form atrihydroxyphenyl-linker-active agent conjugate.

In a related aspect, the invention further provides methods ofimmobilizing an active agent on a substrate, including the steps ofdepositing a primer compound on a substrate thereby forming a primedsubstrate, combining in solution a compound including a trihydroxyphenylgroup and an active agent to couple the compound including atrihydroxyphenyl group and the active agent, thereby forming a solutionof an active agent-trihydroxyphenyl conjugate, and contacting the primedsubstrate with the solution of the active agent-trihydroxyphenylconjugate, thereby immobilizing the active agent on the substrate. Thecompound including a trihydroxyphenyl group can be a small molecule or apolymer including a trihydroxyphenyl group. The polymer can be a polymerincluding the trihydroxyphenyl group in the backbone of the polymer, oralternatively a polymer including at least one monomer having a pendanttrihydroxyphenyl group. The active agent-trihydroxyphenyl conjugate caninclude active agents coupled to linker compounds that are furthercoupled to trihydroxyphenyl conjugates. The combining and contactingsteps can be conducted simultaneously such that the compound includingthe trihydroxyphenyl group and the active agent are combined in thepresence of the primed substrate, or alternatively the combining andcontacting steps can be conducted separately and in sequence.

As used herein, “conjugate” refers to the species that result from thecoupling together of two or more of a compound including atrihydroxyphenyl group, a linker compound, and/or an active agent. Thespecies that have been conjugated are provided immediately before theterm “conjugate.” The conjugate can be formed by coupling the twospecies that are to form a conjugate, as defined above.

In a related aspect, the invention further provides methods ofimmobilizing an active agent on a substrate surface, including the stepsof, depositing a primer compound on a substrate, thereby forming aprimed substrate, contacting the primed substrate with a solution ofgallic acid to couple the trihydroxyphenyl group of the gallic acid tothe primer compound, thereby forming a gallic acid-treated primedsubstrate, and contacting the gallic acid-treated primed substrate witha solution of an active agent to couple the active agent to thetrihydroxyphenyl group of the gallic acid, thereby immobilizing theactive agent on the substrate surface. The method can further includethe step of contacting the gallic acid-treated primed substrate with asolution of a linker compound thereby coupling the linker compound tothe trihydroxyphenyl group and/or the primer compound of the gallicacid-treated primed substrate, prior to contacting thetrihydroxyphenyl-treated substrate with the solution of active agent.

In another related aspect, the invention provides substrates having anactive agent immobilized on a surface thereof, the substrate including alayer of a primer compound on the substrate surface, wherein the layerof the primer compound includes a trihydroxyphenyl group coupledthereto, and wherein the trihydroxyphenyl group has an active agentcoupled thereto and thereby immobilized on the substrate surface. Thecompound including a trihydroxyphenyl group can be a small molecule or apolymer including a trihydroxyphenyl group. The polymer can be a polymerincluding the trihydroxyphenyl group in the backbone of the polymer, oralternatively a polymer including at least one monomer having a pendanttrihydroxyphenyl group. The active agent can be coupled to thetrihydroxyphenyl group and/or the primer compound via a linker compound,so as to immobilize the active agent on the substrate.

In another related aspect, the invention provides medical devicesincluding a substrate according to the invention. Medical devices andmedical device components comprising substrates according to theinvention can include active agents that advantageously render thedevice or device component antibacterial, antifouling, and/oranti-thrombogenic. Of course, the active agents can demonstrate othertherapeutic or beneficial activities.

The medical devices and medical device components comprising activeagents immobilized thereto can be particularly advantageous because themedical device or device component can be effectively “coated” byimmobilizing an active agent on/to a (substrate) surface thereof andthereby reduce the need to treat a patient with the (same or similar)active agent. For example, patients whose treatment requires anextracorporeal blood circuit, such as for hemodialysis, apheresis, orcoronary bypass, are often administered heparin (or similar actingactive agents) prior to the procedure so as to prevent blood clotformation in the blood circuit pumps and tubings. However, in additionto inhibiting clot formation, administration of significant amounts ofheparin can render the patient susceptible to bleeding after thetreatment. Therefore, it would be advantageous to use blood circuitdevices with heparin immobilized thereto, thereby reducing the amount ofheparin needed for treatment prior to the procedure and the attendantrisk of the patient experiencing bleeding problems and/or needingextended hospitalization or medical care subsequent to the procedure.

In general, the methods according to the invention result in an activeagent immobilized on a substrate surface through the use of a compoundincluding a trihydroxyphenyl group that can couple to a primer layercoupled to and/or deposited on the substrate surface. The methodsdescribed herein can include the use of solutions and plasmas of primercompounds, solutions of compounds including a trihydroxyphenyl group(e.g., solutions of trihydroxyphenyl-linker conjugates, solutions ofsmall molecule compounds including trihydroxyphenyl groups such asgallic acid, and solutions of polymers including trihydroxyphenyl groupssuch as polygallic acid), solutions of linker compounds, solutions ofactive agents (including solutions of active agent-linker conjugates),and solutions of active agent-trihydroxyphenyl conjugates. The solventsused to prepare the solutions of primer compounds, solutions ofcompounds including a trihydroxyphenyl group, solutions of linkercompounds, solutions of active agents, and solutions of activeagent-trihydroxyphenyl conjugates can be any solvent suitable to act asa carrier for the primer compounds, compounds comprising atrihydroxyphenyl group, linker compounds, active agents, and/or activeagent-trihydroxyphenyl conjugates. For example, the solutions describedherein can comprise aqueous solutions, other solvents including but notlimited to, alcohols, diols, organosulfurs such as sulfolane, ethers,such as diethyl ether and tetrahydrofuran, alkanes, aromatics,halocarbons, such as chloroform and dichloromethane, and combinations ofthe foregoing. When the term “solution” is used herein, it is notnecessary that the components contained therein completely dissolve.Thus, as used herein, the term solution encompasses both dispersions inwhich components are dispersed and solutions in which components aresubstantially or even completely dissolved. In general, completedissolution of the component is preferred. Further, as used herein, theterm “solution” includes aerosolized solutions.

In one aspect of the invention, the method of immobilizing the activeagent on the substrate surface, includes the steps of:

(a) contacting a substrate with a primer compound, thereby forming aprimed substrate;(b) contacting the primed substrate with a compound comprising atrihydroxyphenyl group thereby coupling the trihydroxyphenyl group tothe primed substrate to provide a trihydroxyphenyl-treated primedsubstrate; and(c) contacting the trihydroxyphenyl-treated primed substrate with anactive agent to couple the active agent to the trihydroxyphenyl group,thereby immobilizing the active agent on the substrate.

In another aspect of the invention, the method of immobilizing theactive agent on the substrate surface, includes the steps of:

(a) contacting a substrate with a primer compound, thereby forming aprimed substrate;(b) contacting the primed substrate with gallic acid to couple thetrihydroxyphenyl group of the gallic acid to the primer compound,thereby forming a gallic acid-treated primed substrate; and(c) contacting the gallic acid-treated substrate with an active agent tocouple the active agent to the trihydroxyphenyl group, therebyimmobilizing the active agent on the substrate.

In a related aspect, the method of immobilizing an active agent on asubstrate surface, includes the steps of:

(a) depositing a primer compound on the substrate thereby forming aprimed substrate;(b) combining in solution a compound including a trihydroxyphenyl groupand an active agent to couple the trihydroxyphenyl group and the activeagent, thereby forming a solution of an active agent-trihydroxyphenylconjugate; and(c) contacting the primed substrate with the solution of the activeagent-trihydroxyphenyl conjugate, thereby coupling the trihydroxyphenylgroup of the active agent-trihydroxyphenyl conjugate to the primedsubstrate and immobilizing the active agent on the substrate.

In refinements of the aforementioned embodiments, the methods furtherinclude washing the primed substrate with water, thereby forming awashed primed substrate, and optionally flowing an inert gas such asnitrogen over the washed primed substrate prior to contacting the washedprimed substrate with the solution of a compound including atrihydroxyphenyl group and/or gallic acid solution.

In another refinement of the aforementioned embodiments, the methodsfurther include washing the trihydroxyphenyl- and/or gallic acid-treatedprimed substrate with water, thereby forming a washed trihydroxyphenyl-and/or gallic acid-treated primed substrate, and optionally flowing aninert gas such as nitrogen over the washed trihydroxyphenyl- and/orgallic acid-treated primed substrate prior to contacting the washedtrihydroxyphenyl- and/or gallic acid-treated primed substrate with thesolution of active agent.

In yet another refinement of the foregoing embodiments, the methodsfurther include washing the substrate with the active agent immobilizedon a surface thereof with water, thereby forming a washed substrate withthe active agent immobilized on a surface thereof and optionally flowingan inert gas such as nitrogen over the washed substrate with the activeagent immobilized on the surface thereof.

In yet another refinement of the foregoing embodiments, the methodsfurther include the step of contacting the trihydroxyphenyl-treatedprimed substrate with a solution of a linker compound thereby couplingthe linker compound to the trihydroxyphenyl group and/or the primercompound of the trihydroxyphenyl-treated primed substrate, prior tocontacting the trihydroxyphenyl-treated substrate with the solution ofactive agent.

The method can be selected such that the density of the activeagent-trihydroxyphenyl conjugates coupled to the substrate can becontrolled. Without intending to be limited by any particular theory, itis believed that when the trihydroxyphenyl group is coupled to theprimed substrate prior to coupling the active agent to thetrihydroxyphenyl group, the resulting trihydroxyphenyl-treated substratehas a relatively dense covering of trihydroxyphenyl groups coupled tothe substrate. It is further believed that when an activeagent-trihydroxyphenyl conjugate is formed prior to coupling thetrihydroxyphenyl group to the primed substrate, the resulting substratewith an active agent immobilized thereto has a relatively lower densityof active agent-trihydroxyphenyl conjugates coupled to the surface, whencompared to the trihydroxyphenyl-treated substrate prepared prior tocoupling the active agent to the trihydroxyphenyl group. When the activeagent-trihydroxyphenyl conjugate is formed prior to coupling thetrihydroxyphenyl group to the substrate, the conditions can be easilycontrolled by one of ordinary skill in the art such that the coupling ofunsubstituted carbons of the trihydroxyphenyl group to the primedsubstrate is favored over the coupling of any potential binding sitespresent on the active agent or reactive groups on the trihydroxyphenylgroup to the primed substrate.

Substrates

In general, the substrate to which the active agent is (or will be)immobilized can be any substrate. The surface of the substrate can behydrophobic or hydrophilic in nature. Suitable substrates can include,but are not limited to, inorganic oxides (e.g., silicas, materialsconventionally known as glass), ceramics, metals including metal oxides,semiconductors, and/or polymeric substrates. Metal substrates caninclude, but are not limited to, stainless steel, cobalt, titanium,nickel, zirconium, tantalum, chromium, tungsten, molybdenum, manganese,iron, vanadium, niobium, hafnium, aluminum, tin, palladium, ruthenium,iridium, rhodium, gold, silver, platinum, oxides of the foregoing,alloys of the foregoing, and combinations of the foregoing. Suitablepolymer substrates can include, but are not limited to, acrylonitrilebutadiene styrenes, polyacrylonitriles, polyamides, polycarbonates,polyesters, polyetheretherketones, polyetherimides, polyethylenes,polyethylene terephthalates, polylactic acids, polymethyl methacrylates,polypropylenes, polystyrenes, polyurethanes, polyvinyl chloride,polyvinylidene chlorides, polyethers, polysulfones, silicones,polydimethylsiloxanes, polytetrafluoroethylene, polyisoprenes, andblends and copolymers thereof. In one aspect, the substrate has asurface including a suitable reactive moiety ab initio. Substrates ofthe invention also include those that have surfaces that have beenactivated (or modified) in order to facilitate the formation of auniform primer layer. Reactive moieties are useful in that they can beused to covalently bond primer compounds to the substrate surface. Suchreactive moieties, however, need not be present as the primer compoundswill still adsorb to/adhere to/couple to/associate with the substrate inthe absence of reactive moieties on the substrate surface.

The substrate according to the invention can be used to provide one ormore surfaces of a medical device or medical device component. Themedical device or medical device component can be any medical device ormedical device component that may benefit from having an active agentimmobilized on the surface thereof, particularly medical devices whichare in regular contact with the biological fluids of a patient. Medicaldevices or medical device components can include but are not limited toinstruments, apparatuses, implements, machines, contrivances, implants,and components and accessories thereof, intended for use in thediagnosis, cure, mitigation, treatment, or prevention of disease orother condition in humans or other animals, or intended to affect thestructure or any function of the body of humans or other animals.Exemplary medical devices can include, but are not limited to,extracorporeal blood circuit devices such as hemodialysis and coronarybypass pumps and components thereof. Autotransfusion, apheresis,hemofiltration, plasmapheresis, and extracorporeal membrane oxygenationalso involve the use of an extracorporeal blood circuit for removingblood from a patient's circulation and applying a process thereto priorto returning the blood to the patient's circulation.

Specific medical devices and/or medical device components that includesubstrates that benefit from having an active agent immobilized on thesurface thereof include, but are not limited to, tubing; fluid bags;septa; stopcocks; clamps; filters; catheters, such as venous catheters,urinary catheters, Foley catheters, intraurethral catheters,intra-arterial catheters, intraosseous catheters, intrathecal catheters,intra-pulmonary catheters and pain management catheters; tracheal tubes;nasogastric tubes; dialysis sets; dialysis connectors; stents; abdominalplugs; feeding tubes; indwelling devices; surgical tools; needles;cannulae; medical pumps; pump housings; gaskets such as siliconeO-rings; syringes; surgical sutures; filtration devices; drugreconstitution devices; implants; metal screws; and metal plates.Additional exemplary medical devices include, but are not limited to,invasive medical devices, durable medical devices, medical fluidcontainers, medical fluid flow systems, infusion pumps, patientmonitors, and any other medical devices which regularly come intocontact with a patient's biological fluids.

Examples of durable medical devices include intravenous (I.V.) pumps,patient monitors, and the like. Examples of medical fluid flow systemsinclude I.V. sets, intraperitoneal sets, and components thereof, suchas, for example, Luer access devices. A typical I.V. set uses plastictubing to connect a phlebotomized subject to one or more medical fluidsources, such as intravenous solutions or medicament containers. I.V.sets optionally include one or more access devices providing access tothe fluid flow path to allow fluid to be added to or withdrawn from theIV tubing. Access devices advantageously eliminate the need torepeatedly phlebotomize the subject and allow for immediateadministration of medication or other fluids to the subject, as is wellknown. Access devices can be designed for use with connecting apparatusemploying standard Luers, and such devices are commonly referred to as“Luer access devices,” “Luer-activated devices,” or “LADs.” LADs can bemodified with one or more features such as antiseptic indicatingdevices. Various LADs are illustrated in U.S. Pat. Nos. 5,242,432,5,360,413, 5,730,418, 5,782,816, 6,039,302, 6,669,681, and 6,682,509,and U.S. Patent Application Publication Nos. 2003/0141477, 2003/0208165,2008/0021381, and 2008/0021392, the disclosures of which are herebyincorporated by reference in their entireties.

I.V. sets or intraperitoneal sets can incorporate additional optionalcomponents including, for example, septa, stoppers, stopcocks,connectors, protective connector caps, connector closures, adaptors,clamps, extension sets, filters, and the like. Thus, additional suitablemedical devices and medical device components which may be benefit fromthe invention include, but are not limited to: I.V. tubing, I.V. fluidbags, I.V. set access devices, septa, stopcocks, I.V. set connectors,I.V. set connector caps, I.V. set connector closures, I.V. set adaptors,clamps, I.V. filters, I.V. pumps, I.V. poles, catheters, needles,cannulae, stethoscopes, patient monitors, intraperitoneal tubing,intraperitoneal fluid bags, access devices for intraperitoneal sets,intraperitoneal set connectors, intraperitoneal set adaptors, andintraperitoneal filters. Representative access devices include, but arenot limited to: Luer access devices including, but not limited to,needleless Luer access devices. The surface of the medical device can beany substrate as described herein.

Primer Compound

Generally, the primer compound can be any compound that when depositedon a substrate forms a layer on the surface of the substrate and allowsa trihydroxyphenyl group to couple to the primer. As used herein, theterm “primer compound” includes both small molecules and polymers. Thetrihydroxyphenyl group adsorbs to/adheres to/couples to/associates withthe primer compound through covalent bond formation, hydrogen bondformation, ionic bond formation, van der Waals interactions, orcombinations of the foregoing. Typically, the compound including atrihydroxyphenyl group is coupled to the primer by forming one or morecovalent bonds with the primer. In general, the primer compound isbelieved to form a “network” with the compounds including atrihydroxyphenyl group. As used herein, the term “network” refers tocovalent bonds formed between an unsubstituted carbon of thetrihydroxyphenyl group phenyl ring and any two or more compoundsselected from a primer compound, a second trihydroxyphenyl group of acompound including a trihydroxyphenyl group (which may be atrihydroxyphenyl group of a small molecule including a trihydroxyphenylgroup and/or a polymer including a trihydroxyphenyl group), and/orcombinations of the foregoing. In embodiments wherein a relatively highdensity of primer compounds and/or compounds including atrihydroxyphenyl group are covalently bound, a cross-linked network canbe formed. In embodiments wherein there is a relatively low density ofprimer compounds and/or compounds including a trihydroxyphenyl groupcovalently bound, the resulting network may not be cross-linked.

Suitable primer compounds include a nucleophilic group. Suitablenucleophilic groups are well known in the art and can include, but arenot limited to, hydroxyl, alkoxide, amine, nitrite, thiol, thiolate,imidazole, and combinations thereof. Suitable primer compounds include,but are not limited to, oligosaccharides such as chitooligosaccharide,fructooligosaccharide, galactooligosaccharide, mannanoligosaccharide,polyamines such as ethylenediamine, 1,2-diaminopropane,hexamethylenediamine, tetramethylenediamine, pentamethylenediamine,tetraethylmethylenediamine, spermine, spermidine, and polyethyleneamine,poly(methacrylic acid), poly(acrylic acid), poly(glutamic acid),poly(malic acid), amino functionalized silanes includingalkoxyaminosilanes such as aminopropyltriethyoxysilane,aminopropyldiethoxymethylsilane, aminopropyldimethylethoxysilane, andaminopropyltrimethoxysilane, and mercaptosilanes such asmercaptopropyltrimethoxysilane and mercaptopropylmethyldimethoxysilane.Nucleophilic groups on oligosaccharides include amine and hydroxylgroups; nucleophilic groups on polyamines include amine groups. Ofcourse, other nucleophilic groups are possible and the foregoing groupsare only provided to illustrate aspects of the invention.

Because the primer compound can be a small molecule or a polymer, themolecular weight of the primer compound can be suitably varied over alarge molecular weight range. As described below, the molecular weightof the primer compound is typically chosen such that the primer compoundis fully soluble in a chosen solvent (i.e., preferably, without forminga saturated primer compound solution). Alternatively, when a plasma isused to deposit the primer compound on the substrate, the primercompound can be any molecular weight, provided the compound is suitablyvolatile to be dispersed into the vapor phase.

As described with chitooligosaccharide in the examples below, it isbelieved that the primer compound (chitooligosaccharide) can bind to thecompound including a trihydroxyphenyl group through a nucleophile on theprimer compound that covalently binds with an unsubstituted carbon onthe phenyl ring of the trihydroxyphenyl group. Other primer compoundsnecessarily include a similar nucleophilic group as mentioned above andtherefore can bind to the compound including a trihydroxyphenyl group ina similar fashion as chitooligosaccharide. The primer compound can bindto the compound including a trihydroxyphenyl group through othermechanisms as well. For example, an amine present onchitooligosaccharide can form a Schiff base with a compound including atrihydroxyphenyl group that comprises a carbonyl moiety, for example,gallic acid. Other primer compounds having an amine as a nucleophilicgroup can bind to a compound including a trihydroxyphenyl group thatcomprises a carbonyl moiety in a similar fashion.

In embodiments of the invention, the primer compound can also functionas a secondary active agent (such as an antibacterial agent, anantifouling agent, an anti-inflammatory agent, an anti-thrombogenicagent, e.g., an anticoagulation agent, and combinations thereof). Forexample, oligosaccharides such as chitooligosaccharide canadvantageously act as an antibacterial agent in addition to the primaryintended function as primer compound. As another example, chitosan thathas been functionalized with quaternary amine groups can be used as aprimer compound that also advantageously imparts antibacterialproperties to the substrate.

In general, the primer compound forms a substantially uniform layer onthe substrate surface. As used herein, “uniform” refers to theuniformity of the amount/number density of primer compound on thesubstrate surface per unit area of the substrate surface. Typically, theterm refers to substantially contiguous coverage on the substrate.Substantially contiguous coverage refers to the primer compound beingpresent on at least about 20% to about 100% coverage of the substratesurface, for example, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90% and/or about 100% coverage of the substratesurface. Substantially contiguous coverage of the primer compound on thesubstrate advantageously results in substantially regular/controlledspacing of the active agent ultimately immobilized on the substrate. Theuniformity of a given primer compound layer can be confirmed using knowntechniques such as scanning electron microscopy (SEM), atomic forcemicroscopy (AFM), Fourier transform infrared (FTIR) imaging, Ramanimaging, ellipsometric imaging, x-ray photoelectron spectroscopy (XPS)combined with depth profiling, and/or static or dynamic secondary ionmass spectrometry (SIMS) imaging combined with depth profiling. Inaddition, as mentioned above, the primer compound and the compoundincluding a trihydroxyphenyl group can be coupled to the substrate, forexample, by forming a network with one another (e.g., the primercompound and the compound including a trihydroxy group can be introducedin sequence or in combination in the presence of a substrate). Theuniformity of the network layer can also be confirmed using knowntechniques such as scanning electron microscopy (SEM), atomic forcemicroscopy (AFM), Fourier transform infrared (FTIR) imaging, Ramanimaging, ellipsometric imaging, x-ray photoelectron spectroscopy (XPS)combined with depth profiling, and/or static or dynamic secondary ionmass spectrometry (SIMS) imaging combined with depth profiling.

Ranges may be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another embodiment according tothe invention includes from the one particular value and/or to the otherparticular value. Similarly, when particular values are expressed asapproximations, but use of antecedents such as “about,” “at leastabout,” or “less than about,” it will be understood that the particularvalue forms another embodiment.

Without intending to be bound by any particular theory, it is believedthat the uniformity of the primer layer that is deposited on thesubstrate surface depends upon the hydrophobic/hydrophilic nature ofboth the substrate surface and the primer compound, and the duration oftime that the primer layer is exposed to the substrate surface. Forexample, it is believed that a substantially uniform layer of ahydrophilic primer compound will form more quickly on a hydrophilicsurface than on a hydrophobic surface.

A substrate surface can be modified such that thehydrophilic/hydrophobic nature of the substrate is changed prior toexposure of the primer compound to the substrate surface in order tofacilitate the formation of a uniform primer layer. Plasma treatments,including but not limited to, argon or corona treatments, chemicaltreatments, including but not limited to, acid treatments, basetreatments, and the like can be used to activate or modify a non-polar,hydrophobic substrate surface to be more polar/hydrophilic. For example,in one embodiment, a surface may be modified to include a hydroxyl groupby oxidation of the substrate surface. Suitable methods of oxidizingsubstrate surfaces are known in the art and can include, for example,treatment of the substrate surface with any oxidation agent, including,but not limited to hydrogen peroxide, inorganic peroxides,permanganates, including the potassium, sodium, ammonium, and calciumsalts, osmium tetroxide, and combinations of the foregoing. As anotherexample, polyester substrates can be activated or modified to include ahydroxyl group by treating the substrate with an acid treatment, a basetreatment, or an argon plasma. Suitable methods to activate or modifythe substrate to include an amine include treating a polyamide substratewith an acid treatment, a base treatment, or an argon plasma. Suitablemethods to modify the substrate to include a thiol include treating apolythioester substrate with an acid treatment, a base treatment, or anargon plasma. Plasma treatments can be followed by exposing the plasmatreated substrate to a gas to generate reactive moieties. For example,plasmas can be used to generate radicals and then followed to generatereactive moieties by exposure to gases such as oxygen, ammonia, andhydrogen sulfide and thereby generate hydroxyl, amine, and thiol,respectively.

In embodiments of the invention, the primer compound is deposited on thesubstrate surface by contacting the substrate surface with a solution ofprimer compound. Deposition methods can include completely immersing thesubstrate in a solution of primer compound, for example, by dip coating.Alternatively, deposition methods can include spraying or casting asolution of primer compound onto the substrate surface, for example, byspin casting or spraying a solution such as an aerosolized solution. Forsubstrates having an interior lumen, such as tubing, the solution canalso be flowed into the lumen to coat the interior thereof. The solventcan be any solvent that is capable of serving as a carrier for theprimer compound. For example, most frequently water is used as thesolvent, but organic solvents including but not limited to, alcohols,diols, organosulfurs such as sulfolane, ethers, such as diethyl etherand tetrahydrofuran, halocarbons, such as chloroform anddichloromethane, and combinations of the foregoing can be used. Water isgenerally preferred for smaller and/or charged primer compounds, butconventional organic solvents can be used, especially for polymericprimer compounds. In embodiments of the methods disclosed herein, thesolution comprising the primer compound is at a pH in a range of about7.5 to about 9.5, or about 8 to about 9, or about 8.5. The solution ofprimer compound may further include a buffer, including, but not limitedto, N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris), andN-tris(hydroxymethyl)methylglycine (Tricine). Of course, one or morecarbonate, phosphate and other known buffer systems for maintainingrelatively high pH values can also be used.

The invention further provides methods of immobilizing an active agentto a substrate surface, including the steps of, depositing the primercompound on the substrate by plasma deposition. The term “plasma” asused herein, describes a partially or fully ionized gas composed ofions, electrons, and neutral species. For example, a layer of reactiveamine groups can be deposited on the substrate surface using a plasmatreatment comprising allylamine generated by the application ofradiofrequency under slight to moderate vacuum. In this example, theplasma treatment results in radical species forming on the surface ofthe substrate that will initiate radical polymerization of theallylamine on the substrate surface. In addition, radicals formed in thevapor phase of the plasma can interact with radicals formed on thesubstrate surface and couple to the substrate.

Suitable plasmas can be generated from various inert gases and reactivegases, as well as mixtures of inert gases, mixtures of reactive gases,and/or mixtures of inert gases and reactive gases. Plasmas for use inaccordance with the present methods can be generated by various knownmethods, such as by the application of electric and/or magnetic fields.Various types of power sources can be used to generate suitable plasmasfor use in the disclosed methods; typical power sources include directcurrent (DC), radiofrequency (RF), microwave, and laser power sources. Aparallel-plate plasma source, for example, uses a RF power source togenerate plasma through gas discharge. Another example of an RF powersource is an inductive coupling plasma source which uses an inductivelycoupled RF source to generate plasma. The RF power source can operate at13.56 MHz or at another suitable frequency readily determined by one ofordinary skill Microwave power sources include, for example, theelectron cyclotron resonance (ECR) source. The microwave frequency canbe 2.45 GHz or another suitable frequency readily determined by one ofordinary skill.

Plasmas can be generated at various pressures, and suitable plasmapressures can be readily determined by one of ordinary skill Plasma canbe generated, for example, at atmospheric pressure or under vacuum.Damage to the substrate can be more prevalent at higher pressurescompared to lower pressures. Thus, the use of lower pressures canprevent or reduce damage to the substrate, thereby allowing increasedexposure times and/or increased power levels to be used. Typicalpressures at which plasma can be generated include pressures of about0.001 Torr to about 760 Torr, for example, about 0.01 Torr to about 100Torr, about 0.05 Torr to about 50 Torr, and/or about 0.1 Torr to about10 Torr, but higher and lower pressures also can be used.

In a further embodiment of the invention, the substrate surface can bemodified to include a radical as a reactive moiety by UV irradiationand/or heat treatment (for example, at about 40 to about 110° C.) of thesubstrate in the presence of an initiator to create radicals on thesurface of the substrate. The initiator can be any initiator known inthe art capable of forming a radical when subjected to UV irradiationand/or elevated temperatures, for example, between about 40 and about110° C. Suitable initiators can include, but are not limited to,benzophenone, peroxides, including but not limited to hydrogen peroxide,benzoyl peroxide, acetyl peroxide, lauryl peroxide, t-butyl peracetate,t-butyl hydroperoxide, and di-t-butyl peroxide, nitrogen dioxide,azobisisobutyronitrile (AIBN), and 2,2-dimethoxy-2-phenylacetophenone(DMPA). A radical generated on the substrate surface can be converted toreactive moieties such as hydroxyl, amine, and thiol by exposure togases such as oxygen, ammonia, and hydrogen sulfide, respectively.

Once a substrate surface has been modified to include a reactive moietysuch as a hydroxyl, the substrate surface can be further modified suchthat one reactive moiety is replaced with a different reactive moiety.For example, a thiol can be replaced by a hydroxyl, or vice versa.

The primer compound adsorbs to/adheres to/couples to/associates with thesubstrate surface through covalent bond formation, hydrogen bondformation, ionic bond formation, van der Waals interactions, orcombinations of the foregoing; when a reactive moiety is present on thesubstrate surface, the primer compound advantageously adsorbs to/adheresto/couples to/associates with the reactive moiety. The primer compoundis further coupled to a compound including a trihydroxyphenyl groupwhich is further coupled to an active agent (either before or aftercoupling of the compound including a trihydroxyphenyl group to thesubstrate) through an unsubstituted carbon on the trihydroxyphenyl groupor through a reactive group on the trihydroxyphenyl group, ultimatelyforming a substrate with an active agent immobilized thereto. Thecompound including a trihydroxyphenyl group adsorbs to/adheresto/couples to/associates with the active agent through covalent bondformation, hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, the compoundincluding a trihydroxyphenyl group is coupled to the active agent byforming one or more covalent bonds with the active agent.

When the primer compound is deposited on the substrate surface bycontacting the substrate surface with a solution of primer compound, theconcentration of the primer compound in the solution of primer compoundcan generally be any concentration. The concentration is typicallychosen such that the primer compound is fully soluble in a chosensolvent, without forming a saturated primer compound solution. Anotherconsideration is the duration of time for conducting the deposition isgenerally less when higher concentrations are used. Exemplary primercompound concentrations can be in a range of about 0.0001 to about 100mg/ml, about 0.001 to about 100 mg/ml, about 0.01 to about 100 mg/ml,about 0.05 to about 100 mg/ml, 0.0001 to about 90 mg/ml, about 0.0001 toabout 80 mg/ml, about 0.0001 to about 70 mg/ml, about 0.0001 to about 60mg/ml, about 0.0001 to about 50 mg/ml, about 0.001 to about 50 mg/ml,about 0.001 to about 40 mg/ml, about 0.001 to about 30 mg/ml, about 0.01to about 30 mg/ml, about 0.01 to about 20 mg/ml, about 0.01 to about 15mg/ml, about 0.01 to about 10 mg/ml, about 0.01 to about 5 mg/ml, about0.05 to about 5 mg/ml, about 0.05 to about 3 mg/ml, about 0.05 to about2 mg/ml, and/or about 0.1 to about 1.5 mg/ml, for example, about 0.1mg/ml, and/or about 1 mg/ml.

The substrate surface can be contacted with and/or immersed in thesolution of primer compound for any duration of time suitable to depositthe primer compound on the substrate surface with the desired primercompound density. The rate of the deposition of the primer compound onthe substrate can depend, in part, on the concentration of the primercompound in the primer compound solution, the substrate surface tosolution volume ratio, the ionic strength of the solution, the pH of thesolution, and the temperature. The duration of contact of the substratewith the solution of primer compound can be varied for any suitable timeperiod which ultimately provides a layer on a substrate, for example,from about 10 seconds to about 24 hours when using dip coating. When theduration of contact of the substrate with the solution of primercompound increases above 24 hours (and one of the foregoing exemplaryconcentrations of the primer compound is used), little difference in theamount of primer compound deposited and the uniformity of the primerlayer are expected (relative to a 24 hour exposure time). Withoutintending to be bound by theory, while it is believed that after 24hours deposition of the primer compound may continue, it is expectedthat the amount of primer compound deposited after 24 hours will havelittle effect on the amount of active agent that is ultimatelyimmobilized on the substrate surface.

In another embodiment, a primer compound, for example, a polyamine isdeposited on the substrate surface by a plasma treatment. The substratesurfaces can be exposed to the plasma for various periods of time. Thelength of plasma exposure can be readily determined by one of ordinaryskill and confirmed using the spectroscopic techniques for determiningthe uniformity of a primer compound layer mentioned above. Further, thelength of exposure can vary depending on the reactivity of the plasma.Damage to the substrate can be more prevalent after longer exposuretimes compared to shorter exposure times. Thus, the use of shorterexposure times can prevent or reduce damage to the substrate, therebyallowing increased pressure and/or increased power levels to be used.Typically, the substrate surface is exposed for about 1 second to about2 hours, but shorter and longer exposure periods can be used. Generally,the substrate surface is exposed to the plasma for about 5 seconds toabout 1 hour, about 10 seconds to about 30 minutes, about 30 seconds toabout 20 minutes, and/or about 1 minute to about 10 minutes.

The substrate surfaces can be exposed to the plasma for a continuousperiod of time. The substrate surfaces also can be exposed to the plasmafor intermittent (or “pulsed”) periods of time, i.e., the plasmadeposition process can comprise exposure of the substrate surface to theplasma for a period of time, followed by a period during which thesubstrate surface is not exposed to the plasma. Such periods of exposureand non-exposure can be repeated multiple times. Damage to the substrateor substrate coating can be more prevalent after continuous exposureprocesses compared to pulsed exposure processes. Thus, the use of pulsedexposure processes can prevent or reduce damage to the substrate orsubstrate coating, thereby allowing increased pressure and/or increasedpower levels to be used. Increased power levels over pulsed periods mayadvantageously reduce the amount of time in which the substrates areexposed to the plasma, thereby reducing part cycle time and increasingmanufacturing efficiencies.

Compound Including a Trihydroxyphenyl Group

The primed substrate is exposed to a compound including atrihydroxyphenyl group in order to couple the trihydroxyphenyl group tothe primed substrate. As previously described, the compound including atrihydroxyphenyl group adheres/couples to/associates with the primercompound through covalent bond formation, hydrogen bond formation, ionicbond formation, van der Waals interactions, or combinations of theforegoing. Typically, the trihydroxyphenyl group is coupled to theprimer by forming one or more covalent bonds with the primer. Asdescribed above, the compound including a trihydroxyphenyl groupencompasses small molecule compounds, polymers includingtrihydroxyphenyl groups, and trihydroxyphenyl-linker conjugates. Thepolymers including trihydroxyphenyl groups include polymers wherein thetrihydroxyphenyl group is in the polymer backbone as well as polymersincluding pendant trihydroxyphenyl groups. The trihydroxyphenyl-linkerconjugates include small molecule or polymer compounds including atrihydroxyphenyl group coupled to a linker compound.

Generally, suitable trihydroxyphenyl groups have at least two sites ofreactivity such that the trihydroxyphenyl group can bind to a reactivemoiety presented by/on/within the primer layer, thereby forming a primercompound-trihydroxyphenyl group network, and also to at least one of theactive agent, another compound including a trihydroxyphenyl group, alinker compound, and/or combinations of the foregoing. Suitable smallmolecule compounds including a trihydroxyphenyl group include, but arenot limited to, gallic acid, phloroglucinol, carboxylic acid, gallamide,5-methyl-benzene-1,2,3-triol, 3,4,5-trihydroxybenzaldehyde,2,3,4-trihydroxybenzaldehyde, gallacetophenone,3,4,5-trihydroxybenzamide, 2,3,4-trihydroxybenzoic acid,5-hydroxydopamine hydrochloride, methyl gallate, pyrogallol, derivativesthereof and salts of the foregoing. The aforementioned small moleculecompounds can also be used to prepare polymers comprisingtrihydroxyphenyl groups. Gallic acid, through at least the twounsubstituted carbons on its trihydroxyphenyl group phenyl ring is ableto bind to two of a primer compound, an active agent, another gallicacid, a linker compound, and combinations of the foregoing, therebyimmobilizing the active agent on the substrate surface. Gallic acid isalso able to bind to a primer compound, an active agent, another gallicacid, or a linker compound via its carboxylic acid moiety, as describedbelow for a linker compound. Thus, gallic acid advantageously has threehydroxyls as well as three sites of reactivity that may participate inand facilitate the immobilization of an active agent on the substratesurface. Other compounds including a trihydroxyphenyl group necessarilyinclude at least two sites of reactivity, for example, at least twounsubstituted carbons on the phenyl ring and/or reactive groups (such asthe aforementioned carboxylic acid moiety) in order to also be able tocouple to two of a primer compound, an active agent, another compoundincluding a trihydroxyphenyl group, a linker compound, and combinationsof the foregoing, thereby immobilizing an active agent on the substratesurface. Suitable reactive groups on the phenyl ring of thetrihydroxyphenyl group include, but are not limited to, carboxyls,carboxylates, amides, acyl halides, aldehydes, ketones, and esters.

Linker Compounds

The compound including a trihydroxyphenyl group can be coupled to alinker compound thereby forming a trihydroxyphenyl-linker conjugate. Thecompound including a trihydroxyphenyl group adsorbs to/adheresto/couples to/associates with the linker compound through covalent bondformation, hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, thetrihydroxyphenyl group is coupled to the linker compound by forming acovalent bond with the linker compound through an unsubstituted carbonon the trihydroxyphenyl group or through a reactive group on thetrihydroxyphenyl group. The reactive group on the trihydroxyphenyl groupcan be any reactive group that can react with a nucleophile on a linkercompound. Suitable reactive groups on the trihydroxyphenyl groupinclude, but are not limited to, carboxyls, carboxylates, amides, acylhalides, aldehydes, and esters. The reactive group on thetrihydroxyphenyl group can couple to the linker compound, for example,by transesterification or transamidification. The transesterification ortransamidification can optionally be promoted by an activator compoundsuch as N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBt), or1-hydroxy-7-azabenzotriazole (HOAt). Of course, like the linkercompound, an active agent including a nucleophilic group can also coupleto the reactive group of the trihydroxyphenyl group bytransesterification or transamidification.

The linker compound can be any suitable compound that has a first endgroup and a second end group that enables the linker to couple to eithera reactive group and/or an unsubstituted carbon of the trihydroxyphenylgroup and to a polymerizable moiety, so as to form a polymerizablemonomer, or to couple to either a reactive group and/or an unsubstitutedcarbon of the trihydroxyphenyl group and to an active agent so as toform a trihydroxyphenyl-linker-active agent conjugate. Polyethyleneglycols, diamines, diols, and dithiols are all useful representativelinker compounds. In one aspect, suitable linker compounds include, butare not limited to, compounds according to formula (I):

wherein n is an integer of at least 1, R is any nucleophilic group,including but not limited to hydroxyl, alkoxide, amine, nitrite, thiol,thiolate, imidazole, and amino oxy, R″ is R or a reactive groupincluding, but not limited to, carboxyls, carboxylates, amides, acylhalides, aldehydes and esters, and wherein each R′ is the same ordifferent and can be selected from the group consisting of H andsubstituted or unsubstituted lower alkyl, for example C1 to about C5alkyl. When aqueous solutions are used, n is typically about 1 to 5 (aslong as solubility is achieved in the selected aqueous systems); whenorganic solvents are used, n can be about 1 to 10. For example, suitablelinker compounds can include, but are not limited to, linear bis-aminescomprising first and second amine end groups, such as1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, and/or1,6-diaminohexane.

Suitable linker compounds further include any compound that has two ormore terminal functional groups that can couple to either a reactivegroup and/or an unsubstituted carbon of the trihydroxyphenyl group, apolymerizable moiety, and/or an active agent. As used herein, “terminal”refers to the final functional group of any carbon chain or branch,including, the end groups of linear compounds as well as any branch endsof branched compounds. Typically, the functional groups will benucleophiles. Nucleophilic groups are well known in the art and caninclude, but are not limited to, hydroxyl, alkoxide, amine, nitrite,thiol, thiolate, imidazole, aminooxy, and combinations thereof. Forexample, suitable linker compounds can include, but are not limitedbranched polyethylene glycol molecules wherein each branch is terminatedwith a nucleophilic group (including, but not limited to, 8-ArmPEG-aminooxy, 8-Arm PEG-thiol, 8-Arm PEG-amine, 8-Arm PEG-hydroxyl,4-Arm PEG-aminooxy, 4-Arm PEG-thiol, 4-Arm PEG-amine, 4-ArmPEG-hydroxyl, and the like), dithiols, bisamines, and otherpolynucleophiles.

It is believed that, upon contacting in solution a compound including atrihydroxyphenyl group and a linker compound, any reactive group(s)and/or unsubstituted carbons on the trihydroxyphenyl group can couple tothe linker compound thereby forming a trihydroxyphenyl-linker conjugate.The linker compound adsorbs to/adheres to/couples to/associates with thetrihydroxyphenyl group through covalent bond formation, hydrogen bondformation, ionic bond formation, van der Waals interactions, orcombinations of the foregoing. Typically, the linker compound is coupledto the trihydroxyphenyl group by forming one or more covalent bonds withthe trihydroxyphenyl group. Generic trihydroxyphenyl-linker conjugatescan be represented by formula (IIa), (IIb), and (IIc):

wherein X can be halogen, amine, thiol, aldehyde, carboxylic acid,carboxylate, acyl halide, ester, acrylate, vinyl, C1 to C10 branched orlinear alkyl amine, C1 to C10 branched or linear alkyl thiol, C1 to C10branched or linear alkyl aldehyde, C1 to C10 branched or linear alkylcarboxylic acid, C1 to C10 branched or linear alkyl carboxylate, C1 toC10 branched or linear alkyl acyl halide, C1 to C10 branched or linearalkyl ester, or C1 to C10 branched or linear alkyl acrylate and R is alinker compound. With respect to the length of the carbon chains of thelisted substituents, the chain length is typically C1 to C5 when aqueoussolutions are used (as long as solubility is achieved in the selectedaqueous system); when organic solvents are used, the chain length can beC1 to C10. In accordance with compounds (IIa), (IIb), and (IIc) thethree hydroxyl groups can be provided on any three of C₂, C₃, C₄, C₅,and C₆. For example, when the compound including a trihydroxyphenylgroup is carboxylic acid such as gallic acid (and thus X is carboxyl),the trihydroxyphenyl-linker conjugate can be of formula (IIa) or formula(IIb):

wherein, as above, R is the linker compound. Further, when thetrihydroxyphenyl-linker conjugate is a gallic acid-linker conjugateaccording to (IIa) and (IIb), the three hydroxyl groups are provided onC₃, C₄, and C₅, and the linker, R, is provided on the carboxyl group(IIa) or one of C₂ or C₆ (IIb). When the compound including atrihydroxyphenyl group is pyrogallol, the pyrogallol-linker conjugatecan be of formula (IIc):

wherein R is the linker compound and the three hydroxyl groups can beprovided on any consecutive three of C₂, C₃, C₄, C₅, and C₆.

The linker compound can be coupled to a compound including atrihydroxyphenyl group prior to contacting a primed substrate with thecompound including a trihydroxyphenyl group. Alternatively, atrihydroxyphenyl-treated primed substrate may be contacted with asolution of linker compound, thereby coupling the linker compound to thetrihydroxyphenyl group. The linker compound can also couple to theprimer compound (which is already coupled to the compound including atrihydroxyphenyl group), for example, by coupling to the reducing end ofan oligosaccharide. The linker end group that is distal from thetrihydroxyphenyl group can couple to an active agent, thereby forming atrihydroxyphenyl-linker-active agent conjugate, or to a polymerizablemoiety, so as to form a polymerizable monomer.

Polymerizable Monomers/Polymers Having Pendant Trihydroxyphenyl Groups

In embodiments wherein the compound including a trihydroxyphenyl groupis a polymer, the polymer can include at least one monomer having apendant trihydroxyphenyl group. A polymer having a pendanttrihydroxyphenyl group can be polymerized from polymerizable monomersprepared from a small molecule compound including a trihydroxyphenylgroup that has been modified to include a linker compound that includesa polymerizable moiety.

The polymerizable monomer can be formed by coupling a polymerizablemoiety to a trihydroxyphenyl-linker conjugate. Thetrihydroxyphenyl-linker conjugate includes a linker end group distalfrom the trihydroxyphenyl group. The distal end group of the linker canform a covalent bond with a polymerizable moiety.

In general, the polymerizable moiety can be any functional group thatincludes a polymerizable α,β unsaturated end group. Suitablepolymerizable moieties include, but are not limited to, acrylate,methacrylate, acrylamide, methacrylamide, vinyl acetate, and esters ofthe foregoing. The covalent bond between the linker compound and thepolymerizable moiety may be formed by transesterification ortransamidification and may be promoted by an activator compound such asN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole (HOAt).

In some embodiments, a polymerizable monomer including atrihydroxyphenyl group can also be formed by coupling a reactive groupand/or unsubstituted carbon of the phenyl ring of the trihydroxyphenylgroup with a linker compound having a first end group and a second endgroup, wherein the first end group is a nucleophilic group and thesecond end group is a polymerizable α,β unsaturated end group. Suitablelinker compounds of this embodiment include, but are not limited to,2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropylacrylate, 3-hydroxypropyl methacrylate, 4-hydroxybutyl acrylate,4-hydroxybutyl methacrylate, 6-hydroxyhexyl acrylate, 6-hydroxyhexylmethacrylate, N-(3-hydroxy-propyl)methacrylamide,N-(4-hydroxybutyl)acrylamide, N-(4-hydroxybutyl)methacrylamide,N-(6-hydroxyhexyl)-acrylamide, N-(6-hydroxyhexyl)methacrylamide,N-methyl-N-(2-hydroxyethyl)acrylamide,N-methyl-N-(2-hydroxyethyl)methacrylamide,N-methyl-N-(3-hydroxypropyl)acrylamide,N-methyl-N-(3-hydroxypropyl)methacrylamide,N-methyl-N-(4-hydroxybutyl)acrylamide,N-methyl-N-(4-hydroxybutyl)methacrylamide,N-methyl-N-(6-hydroxyhexyl)acrylamide,N-methyl-N-(6-hydroxyhexyl)methacrylamide, and 4-aminobutylacrylamide.Generally, suitable linker compounds of this embodiment can include, butare not limited to, compounds according to formula (III):

wherein n is 0 or an integer of at least 1, R is any nucleophilic group,including but not limited to hydroxyl, alkoxide, amine, nitrite, thiol,and thiolate, and R′ can be selected from the group consisting ofoxygen, NR″, and CR₂″, and each R″ can be the same or different and canbe selected from the group consisting of H, and substituted orunsubstituted lower alkyl, for example C1 to about C5 alkyl.

Generic polymerizable monomers including a trihydroxyphenyl group arerepresented by formula (IVa), (IVb), and (VIc):

X can be halogen, amine, thiol, aldehyde, carboxylic acid, carboxylate,acyl halide, ester, acrylate, vinyl, C1 to C10 branched or linear alkylamine, C1 to C10 branched or linear alkyl thiol, C1 to C10 branched orlinear alkyl aldehyde, C1 to C10 branched or linear alkyl carboxylicacid, C1 to C10 branched or linear alkyl carboxylate, C1 to C10 branchedor linear alkyl acyl halide, C1 to C10 branched or linear alkyl ester,or C1 to C10 branched or linear alkyl acrylate, Y can be a polymerizablemoiety such as acrylate, methacrylate, acrylamide, methacrylamide, vinylacetate, and esters of the foregoing, and R is a linker compound. Withrespect to the length of the carbon chains of the listed substituents,the chain length is typically C1 to C5 when aqueous solutions are used(as long as solubility is achieved in the selected aqueous system); whenorganic solvents are used, the chain length can be C1 to C10. Inaccordance with compounds (IVa), (IVb), and (IVc) the three hydroxylgroups can be provided on any three of C₂, C₃, C₄, C₅, and C₆. Forexample, when the compound including a trihydroxyphenyl group is acarboxylic acid such as gallic acid (and thus X is carboxyl), thepolymerizable monomer including a trihydroxyphenyl group can be offormula (IIa) or formula (IIb):

wherein R is the linker compound, and Y is the polymerizable moiety.Further, when the compound including a trihydroxyphenyl group is gallicacid, the polymerizable monomer according to (IVa) and (IVb) comprisesthe three hydroxyl groups on C₃, C₄, and C₅, and the linker, R, isprovided on the carboxyl group (IVa) or one of C₂ or C₆ (IVb). When thecompound including a trihydroxyphenyl group is pyrogallol, thepolymerizable monomer can be of formula (IVc):

wherein R is the linker compound, Y is the polymerizable moiety, and thethree hydroxyl groups can be provided on any consecutive three of C2,C3, C4, C5, and C6.

The polymerizable monomer including a trihydroxyphenyl group ispolymerized to form a homopolymer or is copolymerized with one or moresecondary polymerizable monomers (including polymerizable groups) toform a polymer containing at least one monomer having pendanttrihydroxyphenyl group. Copolymers containing pendant trihydroxyphenylgroups and one or more secondary polymerizable monomers can bepolymerized to form random copolymers and/or block copolymers, as isknown in the art. Suitable secondary polymerizable monomers can be anymonomer comprising a polymerizable moiety. Secondary polymerizablemonomers may alternatively have a pendant reactive group (i.e., areactive group that will be pendant from the monomer afterpolymerization), including but not limited to N-hydroxysuccinimide,succinimide, and the like, such that when the secondary monomer isincorporated into the polymer containing at least one monomer having apendant trihydroxyphenyl group the pendant reactive group can couple tothe active agent, thereby forming an active agent-trihydroxyphenylconjugate, or the pendant reactive group can couple to the substrate,thereby forming a trihydroxyphenyl-treated substrate.

Suitable radical initiators for initiating polymerization of thepolymerizable monomer having the trihydroxyphenyl group, and optionallya secondary monomer, include, but are not limited to, azo compounds,organic peroxides, and combinations thereof. Suitable azo compoundsinclude, but are not limited to, azobisisobutyronitrile (AIBN), and1,1-azobis(cyclohexanecarbonitrile) (ABCN). Suitable organic peroxidesinclude, but are not limited to, cyclic peroxides, diacyl peroxides,dialkyl peroxides, hydroperoxides, peroxycarbonates, peroxydicarbonates,peroxyesters, and peroxyketals. Suitable cyclic peroxides include, butare not limited to, 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane.Suitable diacyl peroxides include, but are not limited to,di(3,5,5-trimethylhexanoyl) peroxide. Suitable dialkyl peroxidesinclude, but are not limited to,2,5-dimethyl-2,5-di(tert-butylperoxy)hexane;2,5-dimethyl-2,5-di(tert-butylperoxy)hexyne-3; di-tert-amyl peroxide;di-tert-butyl peroxide; and tert-butyl cumyl peroxide. Suitablehydroperoxides include, but are not limited to, tert-Amyl hydroperoxide;and 1,1,3,3-tetramethylbutyl hydroperoxide. Suitable peroxycarbonatesinclude, but are not limited to, tert-butylperoxy 2-ethylhexylcarbonate; tert-amylperoxy 2-ethylhexyl carbonate; and tert-butylperoxyisopropyl carbonate. Suitable peroxydicarbonates include, but are notlimited to, di(2-ethylhexyl)peroxydicarbonates; and di-sec-butylperoxydicarbonates. Suitable peroxyesters include, but are not limitedto, tert-amyl peroxy-2-ethylhexanoate; tert-amyl peroxyneodecanoate;tert-amyl peroxypivalate; tert-amyl peroxybenzoate; tert-amylperoxyacetate; 2,5-dimethyl-2,5-di(2-ethylhexanoylperoxy)hexane;tert-butyl peroxy-2-ethylhexanoate; tert-butyl peroxyneodecanoate;tert-butyl peroxyneoheptanoate; tert-butyl peroxypivalate tert-butyl,peroxydiethylacetate; tert-butyl peroxyisobutyrate;1,1,3,3-tetramethylbutyl peroxy-2-ethylhexanoate;1,1,3,3-tetramethylbutyl peroxyneodecanoate; 1,1,3,3-tetramethylbutylperoxypivalate; tert-butyl peroxy-3,5,5-trimethylhexanoate; cumylperoxyneodecanoate; tert-butyl peroxybenzoate; and tert-butylperoxyacetate. Suitable peroxyketals include, but are not limited to,1,1-di(tert-amylperoxy)cyclohexane; 1,1-di(tert-butylperoxy)cyclohexane;1,1-di(tert-butylperoxy)-3,3,5-trimethylcyclohexane; and2,2-di(tert-butylperoxy)butane.

The optional secondary monomer can be included in a copolymer with themonomer having the trihydroxyphenyl group in an amount of up to about 95mol %, for example, about 0.5 to about 95 mol %, about 0.5 to about 90mol %, about 1 to about 90 mol %, about 1 to about 85 mol %, about 5 toabout 85 mol %, about 5 to about 80 mol %, about 10 to about 80 mol %,about 10 to about 75 mol %, about 15 to about 75 mol %, about 5 to about70 mol %, about 10 to about 70 mol %, about 15 to about 70 mol %, about15 to about 65 mol %, about 20 to about 65 mol %, about 20 to about 60mol %, about 25 to about 60 mol %, about 25 to about 55 mol %, about 30to about 55 mol %, about 30 to about 50 mol %, about 35 to about 50 mol%, about 35 to about 45 mol %, and/or about 35 to about 40 mol %.

Polymers containing a pendant trihydroxyphenyl group, can be terminatedwith a reactive group through which an active agent can couple to thepolymer. The reactive group can be any reactive group as previouslydescribed herein, including, but not limited to, carboxyls,carboxylates, amides, acyl halides, aldehydes, and esters. The reactivegroup can be included in a compound that can act as a chain transferagent in polymerizations. Suitable chain transfer agents with reactivegroups can include, but are not limited to 3-mercaptopropionic acid,isooctyl 3-mercaptopropionate, and combinations of the foregoing.Alternatively, the active agent will couple to the polymer through anunsubstituted carbon on the pendent trihydroxyphenyl groups and,therefore, the chain end of the polymer need not be able to couple tothe active agent.

As described above, compounds including a trihydroxyphenyl group thatare polymers containing at least one monomer having a pendanttrihydroxyphenyl group can be coupled to a further linker compound,thereby forming a trihydroxyphenyl-linker conjugate that can couple toan active agent. The linker compound adsorbs to/adheres to/couplesto/associates with the trihydroxyphenyl group through covalent bondformation, hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, the linkercompound is coupled to the trihydroxyphenyl group by forming one or morecovalent bonds with the unsubstituted carbons of the trihydroxyphenylgroup.

Polymers Having Trihydroxyphenyl Groups in the Backbone

In alternative embodiments wherein the compound including atrihydroxyphenyl group is a polymer, the trihydroxyphenyl group can bein the backbone of the polymer. A polymer having the trihydroxyphenylgroups in the backbone can be polymerized from a small molecule compoundincluding a trihydroxyphenyl group that has at least two sites ofreactivity. Without intending to be bound by any particular theory, itis believed that, the small molecule compounds including atrihydroxyphenyl group can self polymerize from a quinone-like species,shown below, by the formation of covalent bonds between unsubstitutedcarbon atoms in the phenyl rings of two or more adjacenttrihydroxyphenyl groups.

The trihydroxyphenyl groups of the compounds including atrihydroxyphenyl group of the invention are generally considered to bein a pH dependent equilibrium with a quinone-like species when insolution. For example, the equilibrium between gallic acid (Compound A)and the quinone-like species (Compound B) is shown below. It is believedthat the equilibrium favors the trihydroxylated species, Compound A, ata more acidic pH.

After the compound including a trihydroxyphenyl group has come intocontact with the primer layer, the trihydroxyphenyl group can covalentlybind to a reactive moiety presented by/on/within the layer of primercompound through an unsubstituted carbon of the phenyl ring of thetrihydroxyphenyl group, thereby forming a trihydroxyphenyl-treatedsubstrate.

The small molecule compound including a trihydroxyphenyl group can alsoself polymerize in situ to form polymers containing repeat units of thetrihydroxyphenyl group in the polymer backbone. Without intending to bebound by any particular theory, it is believed that the trihydroxyphenylgroup can self polymerize from the quinone-like species by the formationof covalent bonds between unsubstituted carbon atoms in the phenyl ringsof two or more adjacent trihydroxyphenyl groups. Thus, in oneembodiment, the unsubstituted carbon of the phenyl ring to which anactive agent can couple can be the terminal trihydroxyphenyl group of apolymer chain that is coupled to the substrate surface.

Further, when the compound including a trihydroxyphenyl group is apolymer including pendant trihydroxyphenyl groups, it is believed thatthe unsubstituted carbons of the phenyl rings of the pendanttrihydroxyphenyl groups can internally cross-link if in close proximitywith other pendant trihydroxyphenyl groups on the polymer chain or cancross-link multiple polymer chains.

Further still, it is believed that, upon exposure of thetrihydroxyphenyl treated primed substrate to a solution of active agent,any open binding sites on the trihydroxyphenyl group, or linkercompounds thereon, can couple to the active agent, thereby immobilizingthe active agent on the substrate surface. The active agent adsorbs toadheres to/couples to/associates with the trihydroxyphenyl group throughcovalent bond formation, hydrogen bond formation, ionic bond formation,van der Waals interactions, or combinations of the foregoing. Typically,the active agent is coupled to the trihydroxyphenyl group (which isalready coupled to the layer of primer compound) by forming one or morecovalent bonds with the trihydroxyphenyl group. It is believed that thetrihydroxyphenyl group can covalently bind with a reactive group of theactive agent through an unsubstituted carbon atom of the phenyl ring ofthe trihydroxyphenyl group. Further, it is believed that, when thereactive group of the active agent is a nucleophile, the covalent bondbetween the unsubstituted carbon of the phenyl ring of thetrihydroxyphenyl group and the reactive group of the active agent may beformed by Michael addition. As exemplified with heparin below, theactive agent binds to the trihydroxyphenyl group through a nucleophileon the active agent that covalently binds with an unsubstituted carbonon the phenyl ring of the trihydroxyphenyl group. Other active agentsnecessarily include a similar nucleophilic group as described below andtherefore will bind to the trihydroxyphenyl group in a similar fashionas heparin.

The active agent adsorbs to/adheres to/couples to/associates with alinker coupled to the trihydroxyphenyl group through covalent bondformation, hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, the activeagent is coupled to the linker that is coupled to a trihydroxyphenylgroup (which is already coupled to the layer of primer compound) byforming one or more covalent bonds with the reactive end group. When thereactive group of the linker compound that will couple to the activeagent is a reactive group including, but not limited to, carboxyls,carboxylates, amides, acyl halides, aldehydes and esters, it is believedthat the linker is coupled to the active agent through the reactivegroup on the linker and a nucleophilic group on the active agent. It isfurther believed that when the reactive group of the linker that willcouple to the active agent is a nucleophile, the linker can couple to areducing end of an active agent, including but not limited to heparin,chitosan, quaternary chitosan, etc., through a residual reactive groupon the linker compound.

Coupling of a Compound Including a Trihydroxyphenyl Group and a PrimedSubstrate.

In one embodiment of the invention, the compound including atrihydroxyphenyl group is coupled to a primed substrate surface bycontacting the primed substrate surface with a solution of a compoundincluding a trihydroxyphenyl group. The primed substrate can becompletely immersed in the solution of the compound including atrihydroxyphenyl group, for example, by dip coating. Alternatively, asolution of the compound including a trihydroxyphenyl group can besprayed or cast onto the primed substrate, for example, by spin castingor spraying a solution such as an aerosolized solution. For substrateshaving an interior lumen, such as tubing, the solution can be flowedinto the lumen to coat the interior thereof. The solvent can be anysolvent that is capable of serving as a carrier for the compoundincluding a trihydroxyphenyl group. For example, most frequently wateris used, but other solvents including but not limited to, alcohols,diols, ethers, such as diethyl ether and tetrahydrofuran, halocarbons,such as chloroform and dichloromethane, and combinations of theforegoing can be used. In embodiments of the methods disclosed herein,the solution comprising the compound including a trihydroxyphenyl groupis at a pH in a range of about 7.5 to about 9.5, about 8 to about 9,and/or about 8.5 so the equilibrium is not biased toward eitherdirection of the equilibrium as mentioned above. The solution of thecompound including a trihydroxyphenyl group may further include a bufferin order to maintain the pH within the foregoing ranges, including, butnot limited to, N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris),N-tris(hydroxymethyl)methylglycine (Tricine), and combinations thereof.Of course, one or more of citrate, carbonate, lactate, phosphate andother known buffer systems can also be used. Of course, one or more ofcarbonate, phosphate and other known buffer systems for maintainingrelatively high pH values can also be used.

The concentration of the compound including a trihydroxyphenyl group inthe solution thereof can generally be any concentration. Theconcentration is typically chosen such that the compound including atrihydroxyphenyl group is fully soluble in a chosen solvent, withoutforming a saturated solution of the compound including atrihydroxyphenyl group. Further, because the compound including atrihydroxyphenyl group can self-polymerize in situ, the concentration ofthe compound including a trihydroxyphenyl group is typically selectedsuch that the compound including a trihydroxyphenyl group will becoupled to the primed substrate at an acceptable rate, desirably withoutexcessive self-polymerization or cross-linking, and, therefore, gellingof the solution. Exemplary concentrations of compounds including atrihydroxyphenyl group in solution can be in a range of about 0.0001 toabout 100 mg/ml, about 0.001 to about 100 mg/ml, about 0.01 to about 100mg/ml, about 0.05 to about 100 mg/ml, 0.0001 to about 90 mg/ml, about0.0001 to about 80 mg/ml, about 0.0001 to about 70 mg/ml, about 0.0001to about 60 mg/ml, about 0.0001 to about 50 mg/ml, about 0.001 to about50 mg/ml, about 0.001 to about 40 mg/ml, about 0.001 to about 30 mg/ml,about 0.01 to about 30 mg/ml, about 0.01 to about 20 mg/ml, about 0.01to about 15 mg/ml, about 0.01 to about 10 mg/ml, about 0.01 to about 5mg/ml, and/or about 0.05 to about 5 mg/ml, for example, about 1 mg/ml,and/or about 5 mg/ml.

The primed substrate can be contacted with and/or immersed in thesolution of the compound including a trihydroxyphenyl group for anyduration of time suitable for coupling the compound including atrihydroxyphenyl group to the primed substrate. In embodiments of theinvention the duration can be any duration of time suitable for forminga network of the primer compound and the compound including atrihydroxyphenyl group. The rate of the deposition of the compoundincluding a trihydroxyphenyl group on the primed substrate can depend,in part, on the concentration of the compound including atrihydroxyphenyl group in the solution thereof, the substrate surface tosolution volume ratio, the ionic strength of the solution, the pH of thesolution, and the temperature. The duration of contact of the primedsubstrate with the solution of compound including a trihydroxyphenylgroup can be varied for any suitable time period for coupling thecompound including a trihydroxyphenyl group to the primed substrate, forexample, when using dip coating, from about 10 seconds to about 24hours. When the duration of contact of the primed substrate with thesolution of the compound including a trihydroxyphenyl group increasesabove 24 hours (and one of the foregoing exemplary concentrations of thecompound including a trihydroxyphenyl group is used), little differencein the amount of compound including a trihydroxyphenyl group reactedwith the primer compound is expected (relative to a 24 hour exposuretime). Without intending to be bound by theory, while it is believedthat after 24-hours the compound including a trihydroxyphenyl group maycontinue to be coupled to the primed substrate, it is expected that theamount of the compound including a trihydroxyphenyl group provided after24 hours will have little effect on the amount of active agent that isultimately immobilized on the substrate surface, and further, it isbelieved that the likelihood of the compound including atrihydroxyphenyl group self-polymerizing in solution, even at lowconcentrations, increases with time.

In embodiments of the invention where the compound including atrihydroxyphenyl group comprises a trihydroxyphenyl-linker conjugate, atrihydroxyphenyl-linker conjugate is initially formed by coupling thetrihydroxyphenyl group of a small molecule or polymer compound includinga trihydroxyphenyl group with a nucleophile on a linker compound via anunsubstituted carbon or reactive group on the phenyl ring of thetrihydroxyphenyl group, and is typically followed by contacting thesubstrate with a solution of the trihydroxyphenyl-linker conjugate. Thetrihydroxyphenyl-linker conjugate can be formed by combining in solutiona compound including a trihydroxyphenyl group and a linker compound. Thesolution of the compound including a trihydroxyphenyl group and/or thelinker compound can be prepared in any solvent capable of acting as acarrier for the compound including a trihydroxyphenyl group and/or thelinker compound. For example, most frequently water is used, but othersolvents including but not limited to, alcohols, diols, organosulfurssuch as sulfolane, ethers, such as diethyl ether and tetrahydrofuran,alkanes, aromatics, halocarbons, such as chloroform and dichloromethane,and combinations of the foregoing can also be used.

In refinements of the aforementioned embodiment, the solutions ofcompounds including a trihydroxyphenyl group and linker compounds are ata pH in a range of about 7.5 to about 9.5, or about 8 to about 9, orabout 8.5. The solution of compound including a trihydroxyphenyl groupand/or solution of linker compound may further include a buffer in orderto maintain the pH within the foregoing ranges, including, but notlimited to, N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris), andN-tris(hydroxymethyl)methylglycine (Tricine). Of course, one or more ofcarbonate, phosphate and other known buffer systems can also be used.

The concentrations of the compound including a trihydroxyphenyl groupand linker compound in solution can be any concentration. Theconcentrations are typically chosen such that the compound including atrihydroxyphenyl group and/or linker compound are fully soluble in achosen solvent, without forming saturated solutions. Exemplary compoundincluding a trihydroxyphenyl group and/or linker compound concentrationscan be in a range of about 0.0001 to about 100 mg/ml, about 0.001 toabout 100 mg/ml, about 0.01 to about 100 mg/ml, about 0.05 to about 100mg/ml, 0.0001 to about 90 mg/ml, about 0.0001 to about 80 mg/ml, about0.0001 to about 70 mg/ml, about 0.0001 to about 60 mg/ml, about 0.0001to about 50 mg/ml, about 0.001 to about 50 mg/ml, about 0.001 to about40 mg/ml, about 0.001 to about 30 mg/ml, about 0.01 to about 30 mg/ml,about 0.01 to about 20 mg/ml, about 0.01 to about 15 mg/ml, about 0.01to about 10 mg/ml, about 0.01 to about 5 mg/ml, about 0.05 to about 5mg/ml, and/or about 0.05 to about 3 mg/ml, for example, about 1 mg/ml,about 1.5 mg/ml and/or about 3 mg/ml. The ratio of compound including atrihydroxyphenyl group to linker compound can be in a range of about 1:8to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1, about 1:5to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, and/orabout 1:2 to about 2:1, for example about 1:1.

The trihydroxyphenyl-linker conjugate can be coupled to the primedsubstrate by contacting the primed substrate with a solution oftrihydroxyphenyl-linker conjugate. The primed substrate can becompletely immersed in the solution of the trihydroxyphenyl-linkerconjugate, for example, by dip coating. Alternatively, a solution of thetrihydroxyphenyl-linker conjugate can be sprayed or cast onto the primedsubstrate, for example, by spin casting or spraying a solution such asan aerosolized solution. For substrates having an interior lumen, suchas tubing, the solution can be flowed into the lumen to coat theinterior thereof.

The concentration of the trihydroxyphenyl-linker conjugate in thetrihydroxyphenyl-linker conjugate solution can be any concentration. Theconcentration of the trihydroxyphenyl-linker conjugate is typicallychosen such that the trihydroxyphenyl-linker conjugate is fully solublein a chosen solvent, without forming a saturated solution. Exemplarytrihydroxyphenyl-linker conjugate concentrations can be in a range ofabout 0.0001 to about 100 mg/ml, about 0.001 to about 100 mg/ml, about0.01 to about 100 mg/ml, about 0.05 to about 100 mg/ml, 0.0001 to about90 mg/ml, about 0.0001 to about 80 mg/ml, about 0.0001 to about 70mg/ml, about 0.0001 to about 60 mg/ml, about 0.0001 to about 50 mg/ml,about 0.001 to about 50 mg/ml, about 0.001 to about 40 mg/ml, about0.001 to about 30 mg/ml, about 0.01 to about 30 mg/ml, about 0.01 toabout 20 mg/ml, about 0.01 to about 15 mg/ml, about 0.01 to about 10mg/ml, about 0.01 to about 5 mg/ml, about 0.05 to about 5 mg/ml, and/orabout 0.05 to about 3 mg/ml, for example, about 1 mg/ml, about 1.5 mg/mlor about 3 mg/ml.

The primed substrate can be contacted with and/or immersed in thesolution of the trihydroxyphenyl-linker conjugate for any durationsuitable to couple the trihydroxyphenyl-linker conjugate to the primedsubstrate. It is believed that the trihydroxyphenyl-linker conjugate cancouple to the primed substrate through either one or both of the endgroup of the linker compound distal from the trihydroxyphenyl group andany residual reactive groups on the trihydroxyphenyl group. Inembodiments of the invention the duration of contact can be any durationof time suitable for forming a covalent bond between one or more of thelinker or trihydroxyphenyl group with a reactive moiety (when present)on the primed substrate surface. The rate of the coupling of thetrihydroxyphenyl-linker conjugate and the primed substrate can depend,in part, on the concentration of the trihydroxyphenyl-linker conjugatesolution thereof, the substrate surface to solution volume ratio, theionic strength of the solution, the pH of the solution, and thetemperature. The duration of contact of the primed substrate with thesolution of trihydroxyphenyl-linker conjugate can be varied for anysuitable time period for coupling the trihydroxyphenyl-linker conjugateto the primed substrate, for example, when using dip coating, from about10 seconds to about 24 hours. When the duration of contact of the primedsubstrate with the solution of trihydroxyphenyl-linker conjugateincreases above 24 hours (and one of the foregoing exemplaryconcentrations of trihydroxyphenyl-linker conjugate is used), littledifference in the amount of trihydroxyphenyl-linker conjugate coupled tothe primed substrate surface is expected (relative to a 24 hour exposuretime). Without intending to be bound by theory while, it is believedthat the trihydroxyphenyl-linker conjugate may continue to be coupled tothe primed substrate, it is expected that the amount of thetrihydroxyphenyl-linker conjugate provided after 24 hours will havelittle effect on the amount of active agent that is ultimatelyimmobilized on the substrate surface, and further, it is believed thatthe likelihood of the trihydroxyphenyl group self-polymerizing orcross-linking, and therefore gelling of the solution, even at lowconcentrations, increases with time.

Active Agents

The active agent can include, but is not limited to antimicrobialagents, such as antibacterial agents, antifouling agents,anti-inflammatory agents, such as complement inhibitors, including butnot limited to C1 inhibitors, e.g., eculizumab, and C5 inhibitors,anti-thrombogenic agents, such as anti-coagulating agents, andcombinations thereof. For example, the active agent can include, but isnot limited to, chitosan, dextran, linear polyethylene glycol (PEG),looped polyethylene glycol (PEG), polyethylene glycol derivativesincluding, but not limited to thiol-terminated PEG, N-hydroxysuccinimide(NHS)-terminated PEG and amine-terminated PEG, poly(N-vinylpyrrolidone)(PVP) and PVP derivatives including, but not limited to,thiol-terminated PVP, amine-terminated PVP, and carboxyl-terminated PVP,heparin, fractionated heparin, and unfractionated heparin, and heparinderivatives, said heparin derivatives including but not limited to,enoxaparin, dalteparin, and tinzaparin, quaternary ammonium polymers,albumin, polyethylenimine, 4-hydroxycoumarin derivatives such aswarfarin, coumatetralyl, phenprocoumon, acenocoumarol, dicoumarol,tioclomarol, and brodifacoum, and combinations of the foregoing. Inembodiments comprising polyethylene glycol, chitosan, or heparin, themolecular weight can be in a range of about 500 Da to about 1,000,000Da, or about 1000 Da to about 500,000 Da, about 2000 Da to about 500,000Da, about 2000 Da to about 250,000 Da, and/or about 2000 Da to about100,000 Da. In general, the active agent includes a functional group.Suitable functional groups include, but are not limited to, nucleophilicgroups. Nucleophilic groups are well known in the art and can include,but are not limited to, hydroxyl, alkoxide, amine, nitrite, thiol,thiolate, imidazole, and combinations thereof. Nucleophilic groups onchitosan include amine and hydroxyl groups; nucleophilic groups on PEGand/or PEG derivatives include hydroxyl groups, thiol groups, aminegroups; reactive groups on PVP derivatives include carboxyl groups,thiol groups, amine groups; nucleophilic groups on heparin and heparinderivatives include hydroxyl, carboxylate, and sulfate. The thiol,amine, and carboxyl-terminated PVP derivatives can be prepared byterminating PVP polymerization with an appropriate chain transfer agentsuch as, for example, mercaptoacetic acid or mercaptoethylamine, or byfurther derivatizing a carboxyl-terminated PVP such as, for example, byreacting the carboxyl-terminated PVP with cysteamine followed by areducing agent such as tris(2-carboxyethyl)phosphine (TCEP) ordithiothreitol (DTT).

It is believed that, upon exposure of the trihydroxyphenyl-treatedsubstrate to an active agent, any sites of reactivity available on thetrihydroxyphenyl group (i.e., reactive groups and/or unsubstitutedcarbons on the phenyl ring), or linker compound thereon, can couple tothe active agent, thereby immobilizing the active agent on the substratesurface. The active agent adsorbs to/adheres to/couples to/associateswith the trihydroxyphenyl group through covalent bond formation,hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, the activeagent is coupled to the compound including a trihydroxyphenyl group byforming one or more covalent bonds with an unsubstituted carbon on thetrihydroxyphenyl group or through a reactive group on thetrihydroxyphenyl group. The reactive group on the trihydroxyphenyl groupcan be any reactive group that can react with a nucleophile on an activeagent. Suitable reactive groups on the trihydroxyphenyl group include,but are not limited to, carboxyls, carboxylates, amides, acyl halides,aldehydes, and esters. The reactive group on the trihydroxyphenyl groupcan couple to the linker compound, for example, by transesterificationor transamidification. The transesterification or transamidification canoptionally be promoted by an activator compound such asN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole (HOAt). Inembodiments wherein a covalent bond forms between the active agent andan unsubstituted carbon of the phenyl ring of the trihydroxyphenylgroup, it is believed the covalent bond may be formed by Michaeladdition. For example, the active agent chitosan can couple to atrihydroxyphenyl group through a hydroxyl or amine group on the activeagent that covalently binds with an unsubstituted carbon on the phenylring of the trihydroxyphenyl group. Other suitable active agentsnecessarily include a similar nucleophilic group and therefore can alsocouple to the compound including a trihydroxyphenyl group in a similarfashion as chitosan.

In one embodiment of the invention, the active agent is coupled to thetrihydroxyphenyl-treated primed substrate surface by contacting thetrihydroxyphenyl-treated primed substrate surface with the active agent.The active agent can be provided in solution or, if the active agent isa liquid, the active agent can be provided neat. Thetrihydroxyphenyl-treated primed substrate can be completely immersed inthe solution of the active agent, for example, by dip coating.Alternatively, a solution of primer compound can be sprayed or cast ontothe trihydroxyphenyl-treated primed substrate, for example, by spincasting or spraying a solution such as an aerosolized solution. Forsubstrates having an interior lumen, such as tubing, the solution can beflowed into the lumen to coat the interior thereof.

The active agent solution solvent can be any solvent that is capable ofserving as a carrier for the active agent. For example, most frequentlywater is used, but other solvents including but not limited to,alcohols, diols, organosulfurs such as sulfolane, ethers, such asdiethyl ether and tetrahydrofuran, halocarbons, such as chloroform anddichloromethane, and combinations of the foregoing can also be used. Inone embodiment of the methods disclosed herein, the solution of activeagent is at a pH in a range of about 5.5 to about 8.5, or about 6 toabout 8, or about 7.5, when coupling the active agent to atrihydroxyphenyl group or a linker compound. The solution of the activeagent may further include a buffer in order to maintain the pH withinthe foregoing ranges as is well known in the art. Suitable buffers formaintaining such a pH, include, but are not limited to,N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris),4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), andN-tris(hydroxymethyl)methylglycine (Tricine). Of course, one or more ofcarbonate, phosphate and other known buffer systems for maintainingrelatively higher pH values can also be used.

In alternative embodiments, the trihydroxyphenyl-treated primedsubstrate can be contacted with solutions of active agents having alower pH. For example, the solution of active agent can be at a pH in arange of about 4 to about 5.5, for example about 4.0, about 4.1, about4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8,about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, and/orabout 5.5, when coupling the active agent to a trihydroxyphenyl group ora linker compound. Suitable active agents for coupling at lower pHinclude, but are not limited to, heparin and chitosan. The solution ofthe active agent may further include a buffer in order to maintain thepH within the foregoing ranges as is well known in the art. Suitablebuffers for maintaining such a pH include one or more of acetate,citrate, lactate, phosphate and other known buffer systems can also beused.

The concentration of the active agent in the active agent solution cangenerally be any concentration. The concentration of the active agent istypically chosen such that the active agent is fully soluble in a chosensolvent, without forming a saturated active agent solution. Higherconcentrations are generally preferred to reduce the time needed tocouple the active agent to the trihydroxyphenyl group. Exemplary activeagent concentrations can be in a range of about 0.0001 to about 100mg/ml, about 0.001 to about 100 mg/ml, about 0.01 to about 100 mg/ml,about 0.05 to about 100 mg/ml, 0.0001 to about 90 mg/ml, about 0.0001 toabout 80 mg/ml, about 0.0001 to about 70 mg/ml, about 0.0001 to about 60mg/ml, about 0.0001 to about 50 mg/ml, about 0.001 to about 50 mg/ml,about 0.001 to about 40 mg/ml, about 0.001 to about 30 mg/ml, about 0.01to about 30 mg/ml, about 0.01 to about 20 mg/ml, about 0.01 to about 15mg/ml, about 0.01 to about 10 mg/ml, about 0.01 to about 5 mg/ml, about0.05 to about 5 mg/ml, and/or about 0.05 to about 3 mg/ml, for example,about 1 mg/ml, about 1.5 mg/ml and/or about 3 mg/ml.

The trihydroxyphenyl-treated primed substrate can be contacted withand/or immersed in the active agent or solution of active agent for anyduration of time suitable to couple the active agent and thetrihydroxyphenyl group of the trihydroxyphenyl-treated substrate. Therate of the coupling of the active agent to the trihydroxyphenyl-treatedprimed substrate can depend, in part, on the concentration of the activeagent in the active agent solution, the substrate surface to solutionvolume ratio, the ionic strength of the solution, and the temperature.The duration of contact of the trihydroxyphenyl-treated substrate withthe active agent or solution of active agent can be varied for anysuitable time period for providing a layer on a substrate, for examplewhen using dip coating, from about 10 seconds to about 24 hours. Whenthe duration of contact of the trihydroxyphenyl-treated primed substratewith the solution of active agent increases above 24 hours (and one ofthe foregoing exemplary concentrations of active agent is used), littledifference in the amount of active agent immobilized to the substratesurface is expected (relative to a 24 hour exposure time). Withoutintending to be bound by theory, while it is believed that while theactive agent may continue to be immobilized on thetrihydroxyphenyl-treated primed substrate, it is expected that theamount of the active agent immobilized after 24 hours will have littleeffect on the activity (antibacterial, antimicrobial, etc.) of theresulting substrate having an active agent immobilized thereto.

In embodiments of the invention where the compound including atrihydroxyphenyl group comprises a trihydroxyphenyl-linker conjugate,the active agent can adsorb to/adhere to/couple to/associate with alinker coupled to the trihydroxyphenyl group through covalent bondformation, hydrogen bond formation, ionic bond formation, van der Waalsinteractions, or combinations of the foregoing. Typically, the activeagent is coupled to the linker that is coupled to a trihydroxyphenylgroup by forming one or more covalent bonds with the end group of thelinker compound distal from the trihydroxyphenyl group. When the groupof the linker compound that will couple to the active agent is areactive group including, but not limited to, carboxyls, carboxylates,amides, acyl halides, aldehydes, and esters, it is believed that thelinker is coupled to the active agent through the reactive group on thelinker and a nucleophilic group on the active agent. It is furtherbelieved that when the end group of the linker that will couple to theactive agent is a nucleophile such as hydroxyl, alkoxide, amine,nitrite, thiol, and thiolate, the linker can couple to an active agent,including but not limited to heparin, chitosan, quaternary chitosan,etc.

As described above, “active agent” encompasses active agent-linkerconjugates. In embodiments of the invention where the active agentcomprises an active agent-linker conjugate, an active agent-linkerconjugate is initially formed by coupling a nucleophile on the linkercompound with a reactive group of an active agent or by coupling anucleophile on an active agent with a reactive group on a linkercompound, followed by contacting the trihydroxyphenyl-treated substratewith a solution of the active agent-linker conjugate. The active agentadsorbs to/adheres/couples to/associates with a linker compound throughcovalent bond formation, hydrogen bond formation, ionic bond formation,van der Waals interactions, or combinations of the foregoing. The linkercompound can be any linker compound as previously described herein.Typically, the active agent is coupled to a linker compound by formingone or more covalent bonds with an end group of the linker compound. Itis believed that when the linker couples to the active agent through areactive group including, but not limited to, carboxyls, carboxylates,amides, acyl halides, aldehydes, and esters, the linker is coupled tothe active agent through the reactive group on the linker and anucleophilic group on the active agent. It is further believed that whenthe reactive group of the linker is a nucleophile such as hydroxyl,alkoxide, amine, nitrite, thiol, and thiolate, the linker can couple toa reactive group of an active agent, including but not limited toheparin, chitosan, quaternary chitosan, etc., through the nucleophilicgroup on the linker compound.

The active agent-linker conjugate can be formed by combining in solutiona linker compound and an active agent. The solution of the active agentand/or the linker compound can be prepared in any solvent capable ofacting as a carrier for the active agent and/or the linker compound. Forexample, most frequently water is used, but other solvents including butnot limited to, alcohols, diols, ethers, such as diethyl ether andtetrahydrofuran, halocarbons, such as chloroform and dichloromethane,and combinations of the foregoing can also be used.

In refinements of the aforementioned embodiment, the solution of activeagent and/or linker compound is at a pH in a range of about 5.5 to about9.5, or about 8 to about 9, or about 8.5 or about 6 to about 8, or about7.5. The solution of active agent and/or solution of linker compound mayfurther include a buffer in order to maintain the pH within theforegoing ranges, including, but not limited to,N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris), andN-tris(hydroxymethyl)methylglycine (Tricine). Of course, one or more ofcitrate, carbonate, lactate, phosphate and other known buffer systemscan also be used.

In alternative embodiments, the solutions of active agent and/or linkercompound can be maintained at a lower pH. For example, acetate bufferedsolutions can be used, having a pH in a range of about 4 to about 5.5,about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3,about 5.4, and/or about 5.5, when coupling the active agent to thelinker compound to form the active agent-linker conjugate. Suitableactive agents for coupling to linker compounds at lower pH include, butare not limited to, heparin and chitosan. The solutions of active agentand/or linker compound may further include a buffer in order to maintainthe pH within the foregoing ranges as is well known in the art. Suitablebuffers for maintaining such a pH include one or more of acetate,citrate, lactate, phosphate and other known buffer systems can also beused.

The concentrations of the active agent and linker compound in solutioncan be any concentration. In some embodiments, the active agent can bedirectly added to a solution of the linker compound, without firstforming an active agent solution. In alternative embodiments, the activeagent can be provided to a solution of the linker compound in an activeagent solution. The concentrations are typically chosen such that theactive agent and/or linker compound are fully soluble in a chosensolvent, without forming saturated solutions. Exemplary active agentand/or linker compound concentrations can be in a range of about 0.0001to about 100 mg/ml, about 0.001 to about 100 mg/ml, about 0.01 to about100 mg/ml, about 0.05 to about 100 mg/ml, 0.0001 to about 90 mg/ml,about 0.0001 to about 80 mg/ml, about 0.0001 to about 70 mg/ml, about0.0001 to about 60 mg/ml, about 0.0001 to about 50 mg/ml, about 0.001 toabout 50 mg/ml, about 0.001 to about 40 mg/ml, about 0.001 to about 30mg/ml, about 0.01 to about 30 mg/ml, about 0.01 to about 20 mg/ml, about0.01 to about 15 mg/ml, about 0.01 to about 10 mg/ml, about 0.01 toabout 5 mg/ml, about 0.05 to about 5 mg/ml, and/or about 0.05 to about 3mg/ml, for example, about 1 mg/ml, about 1.5 mg/ml and/or about 3 mg/ml.The ratio of active agent to linker compound can be in a range of about1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1, about1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, and/orabout 1:2 to about 2:1, for example about 1:1.

The active agent-linker conjugate can be coupled to thetrihydroxyphenyl-treated primed substrate by contacting thetrihydroxyphenyl-treated primed substrate with a solution of activeagent-linker conjugate. The trihydroxyphenyl-treated primed substratecan be completely immersed in the solution of the active agent-linkerconjugate, for example, by dip coating. Alternatively, a solution of theactive agent-linker conjugate can be sprayed or cast onto the primedsubstrate, for example, by spin casting or spraying a solution such asan aerosolized solution. For substrates having an interior lumen, suchas tubing, the solution can be flowed into the lumen to coat theinterior thereof.

The concentration of the active agent-linker conjugate in the activeagent-linker conjugate solution can be any concentration. Theconcentration of the active agent-linker conjugate is typically chosensuch that the active agent-linker conjugate is fully soluble in a chosensolvent, without forming a saturated solution. Exemplary activeagent-linker conjugate concentrations can be in a range of about 0.0001to about 100 mg/ml, about 0.001 to about 100 mg/ml, about 0.01 to about100 mg/ml, about 0.05 to about 100 mg/ml, 0.0001 to about 90 mg/ml,about 0.0001 to about 80 mg/ml, about 0.0001 to about 70 mg/ml, about0.0001 to about 60 mg/ml, about 0.0001 to about 50 mg/ml, about 0.001 toabout 50 mg/ml, about 0.001 to about 40 mg/ml, about 0.001 to about 30mg/ml, about 0.01 to about 30 mg/ml, about 0.01 to about 20 mg/ml, about0.01 to about 15 mg/ml, about 0.01 to about 10 mg/ml, about 0.01 toabout 5 mg/ml, about 0.05 to about 5 mg/ml, and/or about 0.05 to about 3mg/ml, for example, about 1 mg/ml, about 1.5 mg/ml and/or about 3 mg/ml.

The trihydroxyphenyl-treated primed substrate can be contacted withand/or immersed in the solution of the active agent-linker conjugate forany duration suitable to couple the active agent-linker conjugate to thetrihydroxyphenyl treated primed substrate. It is believed that theactive agent-linker conjugate can couple to the trihydroxyphenyl-treatedprimed substrate through either or both of the end group of the linkercompound distal from the active agent and residual nucleophilic groupson the active agent. It is further believed that the activeagent-trihydroxyphenyl conjugate can couple to either or both of thetrihydroxyphenyl group and the primer compound of thetrihydroxyphenyl-treated primed substrate. In embodiments of theinvention the duration of contact can be any duration of time suitablefor forming a covalent bond between one or more of the linker or activeagent with one or more of the primer compound and/or thetrihydroxyphenyl group of the trihydroxyphenyl-treated primed substrate.The rate of the coupling of the active agent-linker conjugate to thetrihydroxyphenyl-treated primed substrate can depend, in part, on theconcentration of the active agent-linker conjugate solution thereof, thesubstrate surface to solution volume ratio, the ionic strength of thesolution, the pH of the solution, and the temperature. The duration ofcontact of the trihydroxyphenyl-treated primed substrate with thesolution of active agent-linker conjugate can be varied for any suitabletime period for coupling the active agent-linker conjugate to thetrihydroxyphenyl-treated primed substrate, for example, when using dipcoating, from about, about 10 seconds to about 24 hours. When theduration of contact of the substrate with the solution of activeagent-linker conjugate increases above 24 hours (and one of theforegoing exemplary concentrations of active agent-linker conjugate isused), little difference in the amount of active agent immobilized tothe substrate surface is expected (relative to a 24 hour exposure time).Without intending to be bound by theory, while it is believed that whilethe active agent-linker conjugate may continue to be immobilized on thesubstrate, it is expected that the amount of the active agentimmobilized after 24 hours will have little effect on the activity(antibacterial, antimicrobial, etc.) of the resulting substrate havingan active agent immobilized thereto.

Active Agent-Trihydroxyphenyl Conjugates with Optional Linker

In embodiments of the invention, an active agent-trihydroxyphenylconjugate is initially formed by coupling a nucleophile of the activeagent with a reactive group on the trihydroxyphenyl group or by couplinga nucleophile of the trihydroxyphenyl group with a reactive group of theactive agent, followed by contacting the primed substrate with asolution of the active agent-trihydroxyphenyl conjugate. The activeagent-trihydroxyphenyl conjugate can be formed by combining in solutiona compound including a trihydroxyphenyl group and an active agent. Asdescribed previously, the compound including a trihydroxyphenyl groupincludes trihydroxyphenyl-linker conjugates. Therefore, the activeagent-trihydroxyphenyl conjugate encompasses active agents coupled tolinker compounds that are further coupled to trihydroxyphenyl groups.Active agents coupled to linker compounds can be formed as describedabove for active agent-linker conjugates and compounds includingtrihydroxyphenyl groups coupled to linker compounds can be formed asdescribed above for trihydroxyphenyl-linker conjugates. These can thenbe further reacted with a compound including trihydroxyphenyl group oractive agent, respectively, to form active agent-trihydroxyphenylconjugates. Generic active agent-trihydroxyphenyl conjugates can berepresented by formulae (Va-c) and (VIa-c):

wherein X can be halogen, amine, thiol, aldehyde, carboxylic acid,carboxylate, acyl halide, ester, acrylate, vinyl, C1 to C10 branched orlinear alkyl amine, C1 to C10 branched or linear alkyl thiol, C1 to C10branched or linear alkyl aldehyde, C1 to C10 branched or linear alkylcarboxylic acid, C1 to C10 branched or linear alkyl carboxylate, C1 toC10 branched or linear alkyl acyl halide, C1 to C10 branched or linearalkyl ester, or C1 to C10 branched or linear alkyl acrylate, and R is alinker compound. With respect to the length of the carbon chains of thelisted substituents, the chain length is typically C1 to C5 when aqueoussolutions are used (as long as solubility is achieved in the selectedaqueous system); when organic solvents are used, the chain length can beC1 to C10. In accordance with compounds (Va-c) and (VIa-c), the threehydroxyl groups can be provided on any three of C₂, C₃, C₄, C₅, and C₆.For example, when the compound including a trihydroxyphenyl group is acarboxylic acid such as gallic acid (and thus X is carboxyl), the activeagent-trihydroxyphenyl conjugate can be of formula (Va), (Vb), (VIa), or(VIb):

wherein R is the linker compound and Active Agent denotes an activeagent. Further, when the compound including a trihydroxyphenyl group isgallic acid, consistent with the active agent-trihydroxyphenylconjugates according to (Va), (Vb), (VIa), and (VIb), the three hydroxylgroups are provided on C₃, C₄, and C₅, and the linker is provided on thecarboxyl group (IVa) or one of C₂ or C₆ (IVb). When the compoundincluding a trihydroxyphenyl group is pyrogallol, the activeagent-trihydroxyphenyl conjugate can be of formula (Vc) or (VIc):

wherein R is the linker compound and the three hydroxyl groups can beprovided on any consecutive three of C₂, C₃, C₄, C₅, and C₆.

In embodiments of the invention wherein the active agent is provided asa neat liquid, the active agent can be the solvent in which the activeagent-trihydroxyphenyl conjugate is formed. In embodiments of theinvention wherein a solution of an active agent is combined with asolution of a compound including a trihydroxyphenyl group, the solutionof the active agent and/or the compound including a trihydroxyphenylgroup can be prepared in any solvent capable of acting as a carrier forthe active agent and/or the compound including a trihydroxyphenyl group.For example, most frequently water is used, but organic solventsincluding but not limited to, alcohols, diols, organosulfurs such assulfolane, ethers, such as diethyl ether and tetrahydrofuran, alkanes,aromatics halocarbons, such as chloroform and dichloromethane, andcombinations of the foregoing can also be used.

In refinements of the aforementioned embodiment, the solution of activeagent and/or solution of compound including a trihydroxyphenyl group isat a pH in a range of about 7.5 to about 9.5, or about 8 to about 9, orabout 8.5. The solution of active agent and/or solution of compoundincluding a trihydroxyphenyl group may further include a buffer in orderto maintain the pH within the foregoing ranges, including, but notlimited to, N,N-bis(2-hydroxyethyl)glycine (Bicine),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),tris(hydroxymethyl)methylamine (Tris), andN-tris(hydroxymethyl)methylglycine (Tricine). Of course, one or more ofcitrate, carbonate, lactate, phosphate and other known buffer systemscan also be used. In alternative embodiments, solution of active agentand/or solution of compound including a trihydroxyphenyl group can havea lower pH. For example, acetate buffered solutions can be used fordeposition of active agents at a pH in a range of about 4 to about 5.5,about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3,about 5.4, and/or about 5.5. Suitable active agents for coupling to acompound including a trihydroxyphenyl group in solution at lower pHinclude, but are not limited to, heparin.

The concentrations of the active agent and compound including atrihydroxyphenyl group in solution can generally be any concentration.In some embodiments, the active agent can be directly added to asolution of the compound including a trihydroxyphenyl group, withoutfirst forming an active agent solution. In alternative embodiments, theactive agent can be provided to a solution of the compound including atrihydroxyphenyl group in an active agent solution. The concentrationsare typically chosen such that the active agent and/or compoundincluding a trihydroxyphenyl group are fully soluble in a chosensolvent, without forming saturated solutions. Exemplary active agentand/or compound including a trihydroxyphenyl group concentrations can bein a range of about 0.0001 to about 100 mg/ml, about 0.001 to about 100mg/ml, about 0.01 to about 100 mg/ml, about 0.05 to about 100 mg/ml,0.0001 to about 90 mg/ml, about 0.0001 to about 80 mg/ml, about 0.0001to about 70 mg/ml, about 0.0001 to about 60 mg/ml, about 0.0001 to about50 mg/ml, about 0.001 to about 50 mg/ml, about 0.001 to about 40 mg/ml,about 0.001 to about 30 mg/ml, about 0.01 to about 30 mg/ml, about 0.01to about 20 mg/ml, about 0.01 to about 15 mg/ml, about 0.01 to about 10mg/ml, about 0.01 to about 5 mg/ml, about 0.05 to about 5 mg/ml, and/orabout 0.05 to about 3 mg/ml, for example, about 1 mg/ml, about 1.5 mg/mland/or about 3 mg/ml. The ratio of active agent to compound including atrihydroxyphenyl group can vary depending on if the active agent is asmall molecule or a polymer, as well as if the compound including atrihydroxyphenyl group is a small molecule or a polymer. For example,when the active agent is a polymer and the compound including atrihydroxyphenyl group is a small molecule, one active agent couldcouple thousands of compounds including a trihydroxyphenyl group.Alternatively, when the active agent is a small molecule and thecompound including a trihydroxyphenyl group is a polymer, one compoundincluding a trihydroxyphenyl group could couple to thousands of activeagents. Suitable ratios of active agents to compounds including atrihydroxyphenyl can, therefore, be in a range of about 1:5,000 to about5, 000:1, including all intermediate ranges, such as about 1:5 to about5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, and/or about 1:2 toabout 2:1, for example about 1:1.

The active agent-trihydroxyphenyl conjugate can be coupled to the primedsubstrate by contacting the primed substrate with a solution of activeagent-trihydroxyphenyl conjugate. The primed substrate can be completelyimmersed in the solution of the active agent-trihydroxyphenyl conjugate,for example, by dip coating. Alternatively, a solution of the activeagent-trihydroxyphenyl conjugate can be sprayed or cast onto the primedsubstrate, for example, by spin casting or spraying, using a solutionsuch as an aerosolized solution. For substrates having an interiorlumen, such as tubing, the solution can be flowed into the lumen to coatthe interior thereof.

The concentration of the active agent-trihydroxyphenyl conjugate in theactive agent-trihydroxyphenyl conjugate solution can be anyconcentration. The concentration of the active agent-trihydroxyphenylconjugate is typically chosen such that the activeagent-trihydroxyphenyl conjugate is fully soluble in a chosen solvent,without forming a saturated solution. Exemplary activeagent-trihydroxyphenyl conjugate concentrations can be in a range ofabout 0.0001 to about 100 mg/ml, about 0.001 to about 100 mg/ml, about0.01 to about 100 mg/ml, about 0.05 to about 100 mg/ml, 0.0001 to about90 mg/ml, about 0.0001 to about 80 mg/ml, about 0.0001 to about 70mg/ml, about 0.0001 to about 60 mg/ml, about 0.0001 to about 50 mg/ml,about 0.001 to about 50 mg/ml, about 0.001 to about 40 mg/ml, about0.001 to about 30 mg/ml, about 0.01 to about 30 mg/ml, about 0.01 toabout 20 mg/ml, about 0.01 to about 15 mg/ml, about 0.01 to about 10mg/ml, about 0.01 to about 5 mg/ml, about 0.05 to about 5 mg/ml, and/orabout 0.05 to about 3 mg/ml, for example, about 1 mg/ml, about 1.5 mg/mland/or about 3 mg/ml.

The primed substrate can be contacted with and/or immersed in thesolution of the active agent-trihydroxyphenyl conjugate for any durationof time suitable to couple the trihydroxyphenyl group of the activeagent-trihydroxyphenyl conjugate to the primed substrate. In embodimentsof the invention the duration can be any duration of time suitable forforming a network of the primer compound and the trihydroxyphenyl of theactive agent-trihydroxyphenyl conjugate. The rate of the coupling of theactive agent-trihydroxyphenyl conjugate and the primed substrate candepend, in part, on the concentration of the activeagent-trihydroxyphenyl conjugate in the active agent-trihydroxyphenylconjugate solution, the substrate surface to solution volume ratio, theionic strength of the solution, the pH of the solution, and thetemperature. The duration of contact of the primed substrate with thesolution of active agent-trihydroxyphenyl conjugate can be varied forany suitable time period for coupling the trihydroxyphenyl group withthe primed substrate, for example, when using dip coating, from about 10seconds to about 24 hours. When the duration of contact of the substratewith the solution of active agent-trihydroxyphenyl conjugate increasesabove 24 hours (and one of the foregoing exemplary concentrations ofactive agent-trihydroxyphenyl conjugate is used), little difference inthe amount of active agent immobilized to the substrate surface isexpected (relative to a 24 hour exposure time). Without intending to bebound by theory, while it is believed that while the activeagent-trihydroxyphenyl conjugate may continue to be immobilized on thesubstrate, it is expected that the amount of the active agentimmobilized after 24 hours will have little effect on the activity(antibacterial, antimicrobial) of the resulting substrate having anactive agent immobilized thereto.

The methods, substrates, and medical devices in accordance with theinvention can be better understood in light of the following examples,which are merely intended to illustrate the methods, substrates, andmedical devices and are not meant to limit the scope thereof in any way.

EXAMPLES Example 1 Effect of Heparin Concentration on the Immobilizationof Heparin onto Polysulfone Substrates

An anti-thrombogenic agent, heparin, was immobilized onto polysulfonesubstrates. The effect of heparin concentration on the immobilization ofheparin on a polysulfone substrate was analyzed by varying theconcentration of heparin in the heparin solution. The concentration ofheparin in the heparin solution was either 0.1 mg/mL, 1.0 mg/mL, or 5.0mg/mL. The substrates were prepared as followed.

A polysulfone substrate was immersed in a solution ofchitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolved in10 mM Bicine (pH 8.5). The solution with the polysulfone substrateimmersed therein was mildly agitated at room temperature for 24 hours.The substrate was removed from the solution and rinsed with filtered,distilled water. The resulting primed substrate was immersed in asolution of gallic acid (1 mg/mL), dissolved in 10 mM Bicine (pH 8.5).The gallic acid solution with the primed-substrate immersed therein wasmildly agitated at room temperature for 24 hours. The substrate wasremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate wasimmersed in a solution of heparin (0.1 mg/mL, 1.0 mg/mL, or 5.0 mg/mLheparin) dissolved in 10 mM to 300 mM acetate (pH 4.5), supplementedwith 600 mM NaCl. The solution of heparin with the gallic acid treatedprimed substrate immersed therein was mildly agitated for 24 hours. Thesubstrate was removed from the solution of heparin and rinsed withfiltered, distilled water, resulting in a polysulfone substrate withheparin immobilized on the surface thereof.

Thus, Example 1 illustrates the immobilization of heparin onto apolysulfone substrate according to the invention. Immobilization ofheparin was confirmed using XPS and through Alcian blue staining of theanionic heparin immobilized on the substrate surface.

FIG. 1 shows the x-ray photoelectron spectroscopy survey spectra ofpolysulfone surfaces modified with chitooligosaccharide, gallic acid anddifferent levels of heparin. “PS” is an untreated polysulfone surface,“GA” is a polysulfone substrate with chitooligosaccharide and gallicacid, prepared according to Example 1. “0.1” is a polysulfone substratemodified with chitooligosaccharide, gallic acid and a 0.1 mg/mL heparinsolution, prepared according to Example 1. “1.0” is a polysulfonesubstrate modified with chitooligosaccharide, gallic acid and a 1.0mg/mL heparin solution, prepared according to Example 1. “5.0” is apolysulfone substrate modified with chitooligosaccharide, gallic acidand a 5.0 mg/mL heparin solution, prepared according to Example 1. O1sdesignates oxygen signals, N1s designates nitrogen signals, C1sdesignates carbon signals, and S2s and S2p designate sulfur signals. TheN1s signals are observed on all surfaces including achitooligosaccharide-gallic acid layer. The S1s and S2p peaks are notpresent in the “GA” trace, indicating a layer of chitooligosaccharideand gallic acid has been deposited on the substrate surface. The S1s andS2p peaks present in the “0.1”, “1.0”, and “5.0” traces confirms thatheparin has been immobilized on the substrate.

The elemental composition can be obtained from the XPS analysis and thecompositional data is provided in the following table.

Group C (%) O (%) N (%) S (%) PS 83.6 12.8 0.0 3.6 GA 62.4 32.2 4.9 0.60.1 63.1 30.2 3.6 3.1 1.0 53.0 38.2 5.1 3.7 5.0 62.4 30.7 3.8 3.2

The elemental data further suggests that the amount of heparinimmobilized on a substrate submerged in a heparin solution for 24 hours,as determined by the percentage of sulfur detected, is not stronglydependent on the concentration, in the range tested, of heparin in theheparin solution.

Example 2 Immobilization of Heparin onto Polysulfone Substrate

An anti-thrombogenic agent, heparin, was immobilized onto a polysulfonesubstrate. A polysulfone substrate was immersed in a solution ofchitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolved in10 mM Bicine buffer (pH of 8.4). The solution with the polysulfonesubstrate immersed therein was mildly agitated at room temperature for90 minutes. The substrate was removed from the solution and rinsed withfiltered, distilled water. The resulting primed substrate was immersedin a solution of gallic acid (1.5 mg/mL) in 100 mM Bicine buffer (pH7.6). The gallic acid solution with the primed-substrate immersedtherein was mildly agitated at room temperature for 90 minutes. Thesubstrate was removed from the gallic acid solution and rinsed withfiltered, distilled water. The resulting gallic acid treated primedsubstrate was immersed in a solution of heparin (1 mg/mL) in 0.3 Msodium acetate and 0.6 M sodium chloride solution. The solution ofheparin with the gallic acid treated primed substrate immersed thereinwas mildly agitated for about 12 hours. The substrate was removed fromthe solution of heparin and rinsed with filtered, distilled waterresulting in a polysulfone substrate with heparin immobilized on thesurface thereof. Immobilization of heparin was confirmed using Alcianblue staining of the anionic heparin immobilized on the substrate, asdescribed in Example 10.

Thus, Example 2 illustrates the immobilization of heparin onto apolysulfone substrate according to the invention.

Example 3 Antithrombotic Activity of a Polysulfone Substrate withHeparin Immobilized Thereto

An antithrombogenic agent, heparin, was immobilized onto a polysulfonesubstrate. A polysulfone substrate was immersed in a solution ofchitooligosaccharide primer compound (1.3 mg/mL, 10,000 Mw) dissolved in10 mM Bicine buffer (pH of 8.4). The solution with the polysulfonesubstrate immersed therein was mildly agitated at room temperature for15 minutes. The substrate was removed from the solution and rinsed withfiltered, distilled water. The resulting primed substrate was immersedin a solution of gallic acid (3.5 mg/mL) dissolved in 100 mM Bicinebuffer (pH of 7.7). The gallic acid solution with the primed-substrateimmersed therein was mildly agitated at room temperature for 30 minutes.The substrate was removed from the gallic acid solution and rinsed withfiltered, distilled water. The resulting gallic acid treated primedsubstrate was immersed in a solution of heparin (1.1 mg/mL) in 0.3 Msodium acetate and 0.6 M sodium chloride solution. The solution ofheparin with the gallic acid treated primed substrate immersed thereinwas mildly agitated for 30 minutes. The substrate was removed from thesolution of heparin and allowed to dry, resulting in a polysulfonesubstrate with heparin immobilized on the surface thereof.

The antithrombogenic activity of a polysulfone substrate with heparinimmobilized thereto was evaluated by determining the conversion ofprothrombin to thrombin in a blood sample. The conversion of prothrombinto thrombin in a blood sample containing an uncoated polysulfonesubstrate (designated PS), a gallic acid treated primed controlsubstrate (prepared according to the procedure for forming the gallicacid treated primed substrate in Example 5, designated COS/GA), and asample containing a polysulfone substrate with the active agent heparinimmobilized thereto according to the invention (designated 1 mg/mLHeparin) were determined and compared to the thrombin conversion in acontrol blood sample (designated Blood). As shown in FIG. 2, whenpolysulfone substrates are incubated in blood, the blood samplecomprising the uncoated polysulfone substrate does not demonstratereduced thrombin conversion relative to the control blood sample. Incontrast, the blood sample comprising the polysulfone substrate withheparin immobilized thereto (1 mg/mL Heparin) shows improvedantithrombogenic activity relative to both the sample comprising theuntreated polysulfone substrate and the control blood sample.

F 1+2 was measured using the Siemens Enzygnost® F1+2 (monoclonal) assaykit. The F1+2 fragment is formed during the conversion of prothrombininto active thrombin, during the coagulation cascade. Measurement of theF1+2 fragment allows for quantification of thrombin formed. The F1+2levels were determined by incubating the polysulfone substrates in 2 mLof human blood containing 0.4 U/mL of heparin with slight agitation for2 hours. Aliquots of the blood samples were taken over the 2 hours todetermine the conversion of thrombin over time. The blood samples werethen run according to the Siemens Enzygnost® F1+2 (monoclonal) assay kitpackage insert for determination of F1+2.

Thus, Example 3 demonstrates the immobilization of heparin onto apolysulfone substrate and the retained activity of the heparin after itsimmobilization. While a polysulfone substrate without heparin, PS, wasfouled by thrombin and a gallic acid treated primed control substrate,COS/GA, was fouled by thrombin, the polysulfone substrate treated withheparin, 1 mg/mL Heparin, advantageously demonstrates reduced thrombinconversion relative to PS, thereby confirming the immobilization ofheparin, an antithrombogenic agent, on the substrate surface and theretention of its activity after immobilization on that surface.

Example 4 Antithrombotic Activity of a Polysulfone Substrate withHeparin Immobilized Thereto

An antithrombogenic agent, heparin, was immobilized onto a polysulfonesubstrate. A polysulfone substrate was immersed in a solution ofchitooligosaccharide primer compound (0.1 mg/mL, 10,000 Mw) dissolved in10 mM Bicine buffer (pH of 8.4). The solution with the polysulfonesubstrate immersed therein was mildly agitated at room temperature for10 minutes. The substrate was removed from the solution. The resultingprimed substrate was immersed in a solution of gallic acid (1 mg/mL)dissolved in 100 mM Bicine buffer (pH of 7.7). The gallic acid solutionwith the primed-substrate immersed therein was mildly agitated at roomtemperature for 30 minutes. The substrate was removed from the gallicacid solution and rinsed with filtered, distilled water. The resultinggallic acid treated primed substrate was immersed in a solution ofheparin (5 mg/mL) in 0.3 M sodium acetate and 0.6 M sodium chloridesolution. The solution of heparin with the gallic acid treated primedsubstrate immersed therein was mildly agitated for 30 minutes. Thesubstrate was removed from the solution of heparin and rinsed withfiltered, distilled water, resulting in a polysulfone substrate withheparin immobilized on the surface thereof.

The antithrombogenic activity of a polysulfone substrate heparinimmobilized on was evaluated by determining the conversion ofprothrombin to thrombin in a blood sample. The conversion of prothrombinto thrombin in a blood sample containing an uncoated polysulfonesubstrate (designated PS), a gallic acid treated primed controlsubstrate (prepared according to the procedure for forming the gallicacid treated primed substrate in Example 5, designated COS/GA), and asample containing a polysulfone substrate with the active agent heparinimmobilized thereto according to the invention (designated 5 mg/mLHeparin) were determined and compared to the thrombin conversion in acontrol blood sample (designated Blood). As shown in FIG. 2, whenpolysulfone substrates are incubated in blood, the blood samplecontaining the uncoated polysulfone substrate does not demonstratereduced thrombin conversion. In contrast, the blood sample containingthe polysulfone substrate with heparin immobilized thereon showsimproved antithrombogenic activity relative to both the samplecontaining the untreated polysulfone substrate and the control bloodsample.

F 1+2 was measured using the Siemens Enzygnost® F1+2 (monoclonal) assaykit, as described in Example 3.

Thus, Example 4 demonstrates the immobilization of heparin onto apolysulfone substrate and the retention of the activity of the heparinafter its immobilization. While the polysulfone substrate withoutheparin, PS, was fouled by thrombin and a gallic acid treated primedcontrol substrate, COS/GA, was fouled by thrombin, the polysulfonesubstrate treated with heparin, 5 mg/mL Heparin, advantageouslydemonstrates reduced thrombin conversion relative to PS, therebyconfirming the immobilization of heparin, an antithrombogenic agent, onthe substrate surface and the retention of its activity afterimmobilization on that surface.

Example 5 Antithrombotic Activity of a Polysulfone Substrate withHeparin Immobilized Thereto

An antithrombogenic agent, heparin, was immobilized onto a polysulfonesubstrate. A polysulfone substrate was immersed in a solution ofchitooligosaccharide primer compound (5 mg/mL, 10,000 MW) dissolved in10 mM Bicine (pH 8.4). The solution with the polysulfone substrateimmersed therein was mildly agitated at room temperature for 24 hours.The substrate was removed from the solution. The resulting primedsubstrate was immersed in a solution of gallic acid (2.5 mg/mL)dissolved in 100 mM Bicine (pH 7.7). The gallic acid solution with theprimed substrate immersed therein was mildly agitated at roomtemperature for 20 hours. The substrate was removed from the gallic acidsolution and rinsed with filtered, distilled water. The resulting gallicacid treated primed substrate was immersed in a solution of heparin (1mg/mL) in 0.3 M sodium acetate and 0.6 M sodium chloride solution (pH5.17). The solution of heparin with the gallic acid-treated primedsubstrate immersed therein was mildly agitated for 24 hours. Thesubstrate was removed from the solution of heparin and rinsed withfiltered, distilled water and dried in a laminar flow hood resulting ina polysulfone substrate with heparin immobilized on the surface thereof.

The antithrombogenic activity of a polysulfone substrate with the activeagent heparin immobilized thereto according to the invention wasevaluated by determining the conversion of prothrombin to thrombin in ablood sample. The conversion of prothrombin to thrombin for a bloodsample containing an uncoated polysulfone substrate (designated PS), agallic acid treated primed control substrate (designated COS/GA), and asample containing a polysulfone substrate with the active agent heparinimmobilized thereto (designated 24 h Heparin) were determined. Each ofthe foregoing substrates were compared to the thrombin conversion in acontrol blood sample (designated Blood). The substrates were incubatedin blood and the thrombin conversion was determined. As shown in FIG. 2,the untreated polysulfone substrate and the gallic acid treated primedsubstrate did not demonstrate improved antithrombogenic activity (i.e.,reduced thrombin conversion) relative to the control blood sample,however, the polysulfone substrate having heparin immobilized thereto(24 h Heparin) advantageously demonstrated reduced thrombin conversionrelative to the untreated polysulfone substrate, thereby confirming theimmobilization of heparin, an antithrombogenic agent, on the substratesurface. Further, FIG. 2 shows that a substrate wherein Heparin wasimmobilized thereto using the same concentration of Heparin but ashorter dip time (1 mg/mL, 30 min, Example 3) had similarantithrombogenic activity compared to a substrate wherein Heparin wasimmobilized thereto using a 24 hour dip time (24 h Heparin, Example 5).

Thus, Example 5 shows the immobilization of heparin onto a polysulfonesubstrate and the retention of the activity of the heparin after itsimmobilization.

Example 6 Stability of Heparin Immobilized on a Polysulfone/PolyisopreneSubstrate to Blood and Washing

Multi-component polysulfone and polyisoprene flow cells with heparinimmobilized thereto according to the invention were prepared asdescribed in Example 5 except a mixture of concentrated HCL: 30%H₂O₂.(1:1) was utilized to pretreat the substrate to enhancewettability. The cell was filled with the HCL:peroxide solution andallowed to sit for about 5 min. The cell was then flushed with distilledwater. The heparin was then immobilized to the substrate as described inExample 5. The antithrombogenic activity of a polysulfone/polyisoprenesubstrate with the active agent heparin immobilized thereto according tothe invention was evaluated by visual and microscopic investigation. Theexperiment was set up to compare an unmodified polysulfone/polyisopreneflow cell and a polysulfone/polyisoprene flow cell with heparinimmobilized thereto for thrombus formation. Two blood loops wereassembled with silicone tubing, a blood reservoir and thepolysulfone/polyisoprene flow cell, one loop with the unmodified flowcell and one loop with the heparin modified cell. A roller pump wasutilized to provide a flow rate of 50 mL/min in a continuous loop. Theloop was filled and primed with about 175 mL of blood, which wasrecirculated for 2 hours. The same blood source was utilized for bothloops to remove variability of donors. After completion of therecirculation the loop was rinsed with saline and then washed with aflow of >400 mL/min water heated to 85° C. for a period of no less than60 minutes. The two cells were analyzed and then the process repeatedout to 3 cycles. After completion of the last cycle, optical analysisshowed no visible thrombus on the heparin modified flow cell. Incontrast, the unmodified flow cell had large areas of thrombus visibleto the naked eye. Analysis using SEM showed a very dense fibrinstructure on the unmodified flow cell. In contrast, the heparin modifiedflow cell contained only a small amount of fibrin structure that was notvisible except under high magnification in the SEM.

Thus, Example 6 demonstrates that substrates with the active agentheparin immobilized thereto according to the invention canadvantageously be washed and reused.

Example 7 Immobilization of Heparin onto Polycarbonate Substrate UsingChitooligosaccharide as the Primer Compound

An antithrombogenic agent, heparin, was immobilized onto a polycarbonatesubstrate. A polycarbonate substrate was immersed in a solution ofchitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolved in10 mM Bicine buffer (pH of 8.0). The solution with the polycarbonatesubstrate immersed therein was mildly agitated at room temperature for24 hours. The substrate was removed from the solution and rinsed withfiltered, distilled water. The resulting primed substrate was immersedin a solution of gallic acid (2 mg/mL) in 100 mM Bicine buffer (pH 7.5).The gallic acid solution with the primed-substrate immersed therein wasmildly agitated at room temperature for 24 hours. The substrate wasremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate wasimmersed in a solution of heparin (1 mg/mL) in 0.3 M sodium acetate and0.6 M sodium chloride solution. The solution of heparin with the gallicacid treated primed substrate immersed therein was mildly agitated forabout 24 hours. The substrate was removed from the solution of heparinand rinsed with filtered, distilled water resulting in a polycarbonatesubstrate with heparin immobilized on the surface thereof.

Thus, Example 7 illustrates the immobilization of heparin onto apolycarbonate substrate according to the invention. Immobilization ofheparin was confirmed using Alcian blue staining of the anionic heparinimmobilized on the substrate, as described in Example 10.

Example 8 Immobilization of Heparin onto Polycarbonate Substrate UsingPolyethyleneimine as the Primer Compound

An antithrombogenic agent, heparin, was immobilized onto a polycarbonatesubstrate. A polycarbonate substrate was immersed in a solution ofpolyethyleneimine (PEI) primer compound (1 mg/mL, 10,000 Mw) dissolvedin 10 mM Bicine buffer (pH of 8.0). The solution with the polycarbonatesubstrate immersed therein was mildly agitated at room temperature for24 hours. The substrate was removed from the solution and rinsed withfiltered, distilled water. The resulting primed substrate was immersedin a solution of gallic acid (2 mg/mL) in 100 mM Bicine buffer (pH 7.5).The gallic acid solution with the primed-substrate immersed therein wasmildly agitated at room temperature for 24 hours. The substrate wasremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate wasimmersed in a solution of heparin (1 mg/mL) in 0.3 M sodium acetate and0.6 M sodium chloride solution. The solution of heparin with the gallicacid treated primed substrate immersed therein was mildly agitated forabout 24 hours. The substrate was removed from the solution of heparinand rinsed with filtered, distilled water resulting in a polycarbonatesubstrate with heparin immobilized on the surface thereof.

Thus, Example 8 illustrates the immobilization of heparin onto apolycarbonate substrate according to the invention. Immobilization ofheparin was confirmed using Alcian blue staining of the anionic heparinimmobilized on the substrate, as described in Example 10.

Example 9 Silver Nitrate Test for Confirming the Coupling of theCompound Including a Trihydroxyphenyl Group to the Primer Compound

A variety of compounds including a trihydroxyphenyl group (THP) werecoupled to a primed polysulfone substrate, and the immobilization of theTHP to the substrate was confirmed using a silver nitrate test. Apolysulfone substrate was immersed in a solution of chitooligosaccharideprimer compound (1 mg/mL, 10,000 Mw) dissolved in 10 mM Bicine buffer(pH of 8.0). The solution with the polysulfone substrate immersedtherein was mildly agitated at room temperature for 24 hours. Thesubstrate was removed from the solution and rinsed with filtered,distilled water. The resulting primed substrate was immersed in asolution of one of a compound including a trihydroxyphenyl group (THP),selected from gallic acid (2 mg/mL), pyrogallol (2 mg/mL), or2,4,6-trihydroxybenzaldehyde (2 mg/mL) dissolved in 100 mM Bicine buffer(pH 7.5). The THP solution with the primed-substrate immersed thereinwas mildly agitated at room temperature for 24 hours. The substrate wasremoved from the THP solution and rinsed with filtered, distilled water.The resulting THP treated primed substrate was immersed in a solution of50 mM solution of silver nitrate for about 16 hours, with mildagitation. The substrate was removed from the solution of silver nitrateand rinsed with filtered, distilled water. Any reducing groups on thecompound including a trihydroxyphenyl group would be expected to reducethe silver nitrate if the THP was coupled to the primed substrate. Itwas found that the THP had reduced the silver ions to silvernanoparticles resulting in a brown color to the polysulfone substrate.

Thus, Example 9 corroborates the confirmation of the immobilization ofassorted THP groups onto a polysulfone substrate according to theinvention, via the coupling of THP to chitooligosaccharide whilemaintaining its reactivity.

Example 10 Alcian Blue Test for Confirming the Immobilization of Heparinon a Substrate

An antithrombogenic agent, heparin, was immobilized onto a polycarbonatesubstrate. A polycarbonate substrate was immersed in a solution ofchitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolved in10 mM Bicine buffer (pH of 8.0). The solution with the polycarbonatesubstrate immersed therein was mildly agitated at room temperature for24 hours. The substrate was removed from the solution and rinsed withfiltered, distilled water. The resulting primed substrate was immersedin a solution of gallic acid (2 mg/mL) in 100 mM Bicine buffer (pH 7.5).The gallic acid solution with the primed-substrate immersed therein wasmildly agitated at room temperature for 24 hours. The substrate wasremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate wasimmersed in a solution of heparin (1 mg/mL) in 0.3 M sodium acetate and0.6 M sodium chloride solution. The solution of heparin with the gallicacid treated primed substrate immersed therein was mildly agitated forabout 24 hours. The substrate was removed from the solution of heparinand rinsed with filtered, distilled water. The resulting polycarbonatesubstrate with heparin immobilized on the surface thereof was immersedin a solution of Alcian blue for about 3 hours, with mild agitation. Thesubstrate was removed from the solution of Alcian blue and rinsed withfiltered, distilled water. Any anionic active agents, such as heparin,immobilized on the substrate would be expected to complex with thecationic Alcian blue dye. It was found that the heparin formed a complexwith the Alcian blue, resulting in a blue stain to the polysulfonesubstrate.

Thus, Example 10 illustrates the confirmation of the immobilization ofheparin onto a polycarbonate substrate according to the invention.

Example 11 Immobilization of Polyethylene Glycol on PolysulfoneSubstrate

An antifouling agent, polyethylene glycol (PEG), is immobilized ontopolysulfone substrate. A polysulfone substrate is immersed in a solutionof chitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolvedin 10 mM Bicine (pH 8.4). The solution with the polysulfone substrateimmersed therein is mildly agitated at room temperature for 24 hours.The substrate is removed from the solution and rinsed with filtered,distilled water. The resulting primed substrate is immersed in asolution of gallic acid (2.5 mg/mL) dissolved in 100 mM Bicine (pH 7.7).The gallic acid solution with the primed-substrate immersed therein ismildly agitated at room temperature for 24 hours. The substrate isremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate isimmersed in a solution of NH2-terminated PEG, SH-terminated PEG, and/orNHS-terminated PEG (1 mg/mL, 5,000 MW) in 0.3M sodium acetate and 0.6 Msodium chloride solution (pH 5.17). The solution of PEG with the gallicacid treated primed substrate immersed therein is mildly agitated for 24hours. The substrate is removed from the solution of PEG and rinsed withfiltered, distilled water, resulting in a polysulfone substrate with PEGimmobilized on the surface thereof.

Thus, Example 11 illustrates how the immobilization of polyethyleneglycol onto a polysulfone substrate can be achieved according to theinvention.

Example 12 Immobilization of Polyvinylpyrrolidone on PolysulfoneSubstrate

An antifouling agent, polyvinylpyrrolidone (PVP), is immobilized ontopolysulfone substrate. A polysulfone substrate is immersed in a solutionof chitooligosaccharide primer compound (1 mg/mL, 10,000 Mw) dissolvedin 10 mM Bicine (pH 8.4). The solution with the polysulfone substrateimmersed therein is mildly agitated at room temperature for 24 hours.The substrate is removed from the solution and rinsed with filtered,distilled water. The resulting primed substrate is immersed in asolution of gallic acid (2.5 mg/mL) dissolved in 100 mM Bicine (pH 7.7).The gallic acid solution with the primed-substrate immersed therein ismildly agitated at room temperature for 24 hours. The substrate isremoved from the gallic acid solution and rinsed with filtered,distilled water. The resulting gallic acid treated primed substrate isimmersed in a solution of NH2-terminated PVP (1 mg/mL, 5,000 MW) in 10mM Bicine (pH 8.5). The solution of PVP with the gallic acid treatedprimed substrate immersed therein is mildly agitated for up to 24 hoursat ambient temperature. The substrate is removed from the solution ofPVP and rinsed with filtered, distilled water, resulting in apolysulfone substrate with PVP immobilized on the surface thereof.

Thus, Example 12 illustrates how the immobilization ofpolyvinylpyrrolidone onto a polysulfone substrate can be achievedaccording to the invention.

Of course, other active agents, linker compounds, and compoundsincluding trihydroxyphenyl groups could be used in the foregoingprocedures.

1. A method of immobilizing an active agent on a substrate surface,comprising the steps of: depositing a primer compound on a substrate,thereby forming a primed substrate; contacting the primed substrate witha solution of a compound including a trihydroxyphenyl group, therebyforming a trihydroxyphenyl-treated primed substrate; and contacting thetrihydroxyphenyl-treated primed substrate with a solution of an activeagent, thereby forming a substrate with an active agent immobilized onthe surface thereof.
 2. A method of immobilizing an active agent on asubstrate surface, comprising the steps of: depositing a primer compoundon the substrate thereby forming a primed substrate; combining insolution a compound including a trihydroxyphenyl group and an activeagent, thereby forming a solution of an active agent-trihydroxyphenylconjugate; and contacting the primed substrate with the solution of theactive agent-trihydroxyphenyl conjugate, thereby coupling thetrihydroxyphenyl group of the active agent-trihydroxyphenyl conjugate tothe primed substrate and forming a substrate with an active agentimmobilized on the surface thereof.
 3. The method of claim 1, whereinthe compound including a trihydroxyphenyl group is selected from thegroup consisting of a small molecule comprising a trihydroxyphenyl groupand a polymer comprising a trihydroxyphenyl group.
 4. (canceled)
 5. Themethod of claim 1, wherein the compound including a trihydroxyphenylgroup is selected from the group consisting of gallic acid,phloroglucinol carboxylic acid, gallamide, 5-methyl-benzene-1,2,3-triol,3,4,5-trihydroxybenzaldehyde, 2,3,4-trihydroxybenzaldehyde,gallacetophenone, 3,4,5-trihydroxybenzamide, 2,3,4-trihydroxybenzoicacid, 5-hydroxydopamine hydrochloride, methyl gallate, derivativesthereof, salts of the foregoing, and combinations thereof.
 6. (canceled)7. (canceled)
 8. The method of claim 1, further comprising the step ofcontacting the trihydroxyphenyl-treated primed substrate with a solutionof a linker compound thereby coupling the linker compound to thetrihydroxyphenyl group and/or the primer compound of thetrihydroxyphenyl-treated primed substrate, prior to contacting thetrihydroxyphenyl-treated substrate with the solution of active agent. 9.The method of claim 1, wherein the substrate is selected from the groupconsisting of metal substrates, inorganic oxide substrates, ceramicsubstrates, polymer substrates, semiconductor substrates andcombinations thereof.
 10. (canceled)
 11. (canceled)
 12. (canceled) 13.(canceled)
 14. The method of claim 1, further comprising the step ofmodifying the surface of the substrate prior to contacting the substratewith the solution of the primer compound, wherein the surface of thesubstrate is modified by treating the surface of the substrate with atreatment selected from the group consisting of plasma treatments,corona treatments, and chemical treatments.
 15. (canceled) 16.(canceled)
 17. The method of claim 1, wherein the substrate comprises asurface of a medical device or medical device component.
 18. The methodof claim 17, wherein the medical device comprises an extracorporealblood circuit or components of an extracorporeal blood circuit. 19.(canceled)
 20. (canceled)
 21. The method of claim 1, wherein the primercompound is selected from the group consisting of oligosaccharides,polyamines, amino functionalized silanes, mercaptosilanes, andcombinations thereof.
 22. The method of claim 1, wherein the activeagent is selected from the group consisting of antimicrobial agents,antifouling agents, anti-inflammatory agents, antithrombogenic agents,and combinations thereof.
 23. (canceled)
 24. (canceled)
 25. (canceled)26. The method of claim 1, wherein the active agent is selected from thegroup consisting of chitosan, linear polyethylene glycol, loopedpolyethylene glycol, polyethylene glycol derivatives, fractionatedheparin, unfractionated heparin, heparin derivatives, quaternaryammonium polymers, albumin, polyethylenimine, 4-hydroxycoumarinderivatives, and combinations of the foregoing.
 27. (canceled)
 28. Themethod of claim 1, wherein the solution of the primer compound is at apH in a range of about 7.5 to about 9.5.
 29. (canceled)
 30. (canceled)31. (canceled)
 32. (canceled)
 33. A substrate having an active agentimmobilized on a surface thereof, the substrate comprising a layer of aprimer compound on the substrate surface, wherein the layer of theprimer compound includes a trihydroxyphenyl group coupled thereto, andwherein the compound comprising the trihydroxyphenyl group includes anactive agent coupled thereto, thereby forming a substrate with an activeagent immobilized on the surface thereof.
 34. The substrate of claim 33,wherein the substrate is metal, inorganic oxide, ceramic, semiconductor,or polymeric.
 35. The substrate of claim 33, wherein thetrihydroxyphenyl group is selected from the group consisting of a smallmolecule comprising a trihydroxyphenyl group and a polymer comprising atrihydroxyphenyl group.
 36. (canceled)
 37. The substrate of claim 33,further comprising a linker compound coupled to the trihydroxyphenylgroup and/or the primer compound, wherein the linker compound includesan active agent coupled thereto, thereby forming a substrate with anactive agent immobilized on the surface thereof.
 38. The substrate ofclaim 33, wherein the primer layer on the article has a thickness in arange of about 0.1 nm to about 100 μm.
 39. The substrate of claim 33,wherein the primer is selected from the group consisting ofoligosaccharides, polyamines, amino functionalized silanes, andcombinations thereof.
 40. The substrate of claim 33, wherein the activeagent is selected from the group consisting of antimicrobial agents,antifouling agents, anticoagulants, anti-inflammatory agents,antithrombogenic agents, and combinations of the foregoing. 41.(canceled)
 42. (canceled)
 43. (canceled)
 44. The substrate of claim 33,wherein the active agent is selected from the group consisting ofchitosan, linear polyethylene glycol, looped polyethylene glycol,polyethylene glycol derivatives, fractionated heparin, unfractionatedheparin, heparin derivatives, quaternary ammonium polymers, albumin,polyethylenimine, 4-hydroxycoumarin derivatives, and combinations of theforegoing.
 45. (canceled)
 46. (canceled)
 47. A medical device comprisingthe substrate of claim
 33. 48. The medical device of claim 47, whereinthe medical device comprises an extracorporeal blood circuit. 49.(canceled)
 50. The medical device of claim 47, wherein the medicaldevice comprises tubing, the substrate comprises polysulfone, the primercompound comprises an oligosaccharide, and the active agent comprisesheparin.
 51. (canceled)
 52. (canceled)
 53. (canceled)